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* Departments of Radiation Oncology and Pharmacology and the Ireland Comprehensive Cancer Center, Laboratory of Molecular Stress Responses, Case Western Reserve University, Cleveland, Ohio 44106-4942, USA;
Department of Pharmacology and University of Wisconsin Comprehensive Cancer Center, University of Wisconsin-Madison, Madison, Wisconsin 53792, USA; and
DeRoche Discovery Welwyn, Welwyn Garden City, Hertfordshire AL7 3AY, United Kingdom
1Correspondence: Departments of Radiation Oncology and Pharmacology and the Ireland Cancer Center, Laboratory of Molecular Stress Responses, Case Western Reserve University, 10900 Euclid Ave. (BRB 326), Cleveland, OH 44106-4942, USA. E-mail: dab30{at}po.cwru.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B DNA binding in U1-Mel cells after IR treatment
lasted much longer (i.e., >20 h). U1-Mel cells overexpressing
dominant-negative IB
S32/36A mutant protein were significantly
more resistant to IR exposure and retained both G2/M and
G0/G1 cell cycle checkpoint responses without
significant genetic instability (i.e., polyploid cell populations were
not observed). Nuclear p53 protein levels and DNA binding activity
increased only after high doses of IR (>1200 cGy). Disruption of p53
responses in U1-Mel cells by E6 transfection also abrogated
G0/G1 cell cycle checkpoint arrest responses
and increased polyploidy after IR, but did not alter radiosensitivity.
These data suggest that abrogation of individual components of this
coordinate IR-activated transcription factor response may lead to
divergent alterations in cell cycle checkpoints, genomic instability,
apoptosis, and survival. Such coordinate transcription factor
activation in human cancer cells is reminiscent of prokaryotic SOS
responses, and further elucidation of these events should shed light on
the initial molecular events in the chromosome instability
phenotype.—Yang, C.-R., Wilson-Van Patten, C., Planchon, S. M.,
Wuerzberger-Davis, S. M., Davis, T. W., Cuthill, C.,
Miyamoto, S., Boothman, D. A. Coordinate modulation of Sp1,
NF-kappa B, and p53 in confluent human malignant melanoma cells after
ionizing radiation.
Key Words: PLDR • Sp1 • retinoblastoma control proteins • NF-B • p53 • ionizing radiation • genomic instability • aneuploid/polyploid • apoptosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. An established human
melanoma cell line (U1-Mel) that showed extreme radioresistance
(1)
and high rates of PLDR (potential lethal damage
repair) (3)
has been used as a model system for
investigating resistance to ionizing radiation (IR).
To elucidate mechanism(s) of radioresistance, we previously isolated
three known IR-inducible transcripts (xips) by differential
hybridization [i.e., thymidine kinase (TK), tissue-type plasminogen
activator (t-PA), and DT diaphorase (NQO1)] from confluence-arrested
U1-Mel cells during PLDR responses (4)
. While
investigating the IR activation of transcription factors (TFs) binding
to the TK and t-PA promoters, we discovered that Sp1/RCPs
(retinoblastoma control proteins) binding to an Sp1 consensus sequence
site was enhanced in confluent human normal and neoplastic cells after
IR (5)
. More important, we showed that Sp1/RCP binding
within the TK promoter correlated with the IR induction of TK
transcript and enzyme activity and also with the induction of a number
of immediate early growth response proto-oncogenes (IEGs, such as
c-jun, c-fos, c-myc, egr-1, TGF-ß, TK)
(5
6
7)
. The Sp1 binding site (GGGCGG, i.e., a GC box) is
one type of RCE (retinoblastoma control element). The RCE can be the
target of transcriptional regulation by a series of proteins that are
regulated by the retinoblastoma protein (pRb) (8)
. There
are at least three nuclear RCPs (retinoblastoma control proteins) that
complex with these elements in vitro, and the RCE-RCP
complex may play a role in pRb protein-regulated transcription
(9
10
11)
. The Sp1 protein is only one type of RCP that may
bind and regulate RCE-containing genes, usually causing transcriptional
down-regulation when binding alone (8)
. Sp1-mediated
transcription is thought to be stimulated by phosphorylation via
protein kinases, such as c-abl, but are also regulated by pRb;
transient overexpression of hypophosphorylated pRb can stimulate
Sp1/RCP binding (12)
.
The other known TFs that are activated after IR are p53 (reviewed
in ref 13
) and nuclear factor-kappa B (NF-B) (5
, 14)
. Binding of NF-B to the t-PA promoter (as shown by primer
extension DNase I footprinting) corresponded very closely to the
IR-mediated t-PA transcript induction (4
, 15
, 16)
, but not
to the induction of TK (6)
in U1-Mel cells.
The purpose of this study was to further characterize mechanisms of
coordinate IR activation of Sp1, NF-B, and p53 in confluent U1-Mel
cells under conditions that promote PLDR. Such studies may elucidate a
mammalian equivalent of the prokaryotic SOS response (17)
.
We show that Sp1 and NF-B DNA binding activity increased after low
doses (clinically relevant) of IR. p53 activation, in contrast, did not
occur in confluent U1-Mel cells unless very high doses of IR were used.
The radiosensitivity of U1-Mel cells was considerably altered by
disruption of the regulation of Sp1-pRb (by E7 transfection) or NF-B
(by its cytoplasmic inhibitor, IB S32/36A dominant negative
mutant), but not by disruption of p53 (by E6 transfection). The cell
cycle alterations of E6/E7-transfected U1-Mel cells and dramatically
increased genomic instability (measured as increased polyploid cell
populations) after IR were also observed.
Sp1, IB, and p53 are substrates in vitro for
DNA-dependent protein kinase (DNA-PK) (18
19
20
21)
, which is
required for the repair of DNA double-strand breaks (reviewed in ref
22
). The possibility that DNA-PK is an upstream coordinate
regulator of Sp1, NF-B (via IB, as proposed in refs 19
, 21
), and p53 (as proposed in ref 20
) is also
discussed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 24)
. On confluence arrest, cells were placed in DMEM containing
0.2% FBS overnight (16–18 h) and <6% tritiated thymidine-labeled
nuclei were observed (4
, 23
, 24)
. Cells were treated with
various doses of IR using a 137Cs source (dose
rate: 6 Gy/min). Preparation of nuclear extracts, DNA band shift
assays, and Western blot analyses were performed as described below.
PLDR ability (or enhanced cell survival) was determined by
colony-forming assays (25)
. Briefly, after irradiation,
cells were incubated for 4 h to allow for PLDR, trypsinized,
serially diluted, and then plated in DMEM containing 10% FBS as
described previously (25)
. Experiments were performed at
least three times, each in duplicate. All experiments were performed
using mycoplasma-free cultures. We noted that mycoplasma-contaminated
U1-Mel cells showed increased basal levels and loss of IR induction of
Sp1 and NF-B DNA binding, as well as basal expression of all xips
(4)
.
Stable transfection, measurements of cell cycle distribution, and
apoptosis
Log-phase U1-Mel cells were seeded at low cell density
(1x105 cells/100 mm plate) in DMEM containing
10% FBS, then incubated with retroviral expression systems containing
pLXSN vector alone (vec1), pLXSN-E6, or pLXSN-E7 for control, human
papillomavirus E6, and E7 gene overexpression, respectively. After
4 h, media containing virus were removed and replaced with DMEM
containing 10% FBS. After 48 h, infected cells were selected for
over 2 wk in DMEM containing 500 µg/ml G418 (neomycin). Individual
clones were isolated and maintained in DMEM containing 200 µg/ml
G418. For overexpression of the IB S32/36A mutant protein, U1-Mel
cells were transfected with pCMX vector alone (vec2) or pCMX-IB
S32/36A mutant (26)
, and individual clones were isolated.
IB mutant overexpression was confirmed by Western immunoblot
analyses (data not shown). The wild-type p53 status of nontransfected
and transfected U1-Mel cells was confirmed using SSCP analyses as
described by the manufacturer (Clontech, Inc.) (data not shown). All
transfected U1-Mel clones described above had similar doubling times,
estimated from growth curves to be 27 ± 2 hrs.
At select times post-IR, adherent U1-Mel-vec1, -E6, -E7, -vec2, or
-IB S32/36A cells were harvested by trypsinization. Floating
cells were also collected from media by centrifugation. Adherent and
floating cells were then stained with 50 µg/ml propidium iodide (PI)
in phosphate-buffered saline with 0.5% Nonidet P-40 and RNase A as
described (27)
. Cells were analyzed using a Becton
Dickinson FACStar flow cytometer. Data were analyzed by ModFit LT
(Verity Software House). Apoptotic cells were analyzed as a
sub-G0/G1 (<2N) cell
population after citric acid washing (27)
and apoptosis
was confirmed by evaluating the percentage of apoptotic bodies, using
Hoescht 33238 dye staining and other morphology changes as described
(27)
. The emergence of polyploid cell populations were
grouped and quantified as >4N DNA content. Experiments were performed
at least three times, each in duplicate.
Preparation of nuclear extracts, DNA band shift assays, and
Western immunoblot analyses
Nuclear extracts were prepared at various times and/or
after various doses of IR (5)
. DNA band shift and Western
immunoblot assays were performed as described (5
, 6)
.
Oligomers containing specific DNA binding sites were: Sp1,
5'-GATCGATCGGGGCGGGGCGATC-3'; NF-B,
5'-GATCGAGGGGACTTTCCCTAGC-3'; p53,
5'-GATCCGGACATGCCCGGGCATGTCCG-3'; Oct-1,
5'-GATCGAATGCAAATCACTAGCT-3'. Sp1 (sc-59X) and
p53 (sc-126X, to wild-type p53) antibodies used for supershift assays
were purchased from Santa Cruz Biochemicals (San Diego, Calif.). Oct-1
DNA binding activity remained unchanged after IR (5)
and
was used as loading controls throughout the experiments described above
(data not shown). DNA band shift assays were quantified by
PhosphorImager (Molecular Dynamics) analyses. Western immunoblots of
nuclear extracts were analyzed for alterations in Sp1, p53, and pRb
proteins using the appropriate antibodies [Sp1 (PEP2), p53 (DO-1, HRP
conjugate), and pRb (C-15)] purchased from Santa Cruz Biochemicals.
Nuclear extract (10 µg) from nonirradiated or irradiated (various
doses, various times post-IR) quiescent U1-Mel cells was separated on
8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels,
transferred to immobilon-P membranes (Millipore), probed using
antibodies against p53, Sp1, or pRb (described above), and detected by
enhanced chemiluminescence (ECL, Amersham). All experiments were
repeated at least three times.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B, and p53 in human U1-Mel
cells after IR
. Our goal was to determine
whether Sp1/RCP transcription factor (TF) DNA binding correlated with
IR-inducible endogenous IEG expression, since RCP-dependent regulation
of some IEGs (i.e., c-fos, c-myc, TGF-ß) has been
demonstrated (9
10
11)
. We primarily examined Sp1 consensus
site DNA binding (Figs. 1A, B
),but similar responses were noted using the RCE consensus site (data not
shown). Increases in DNA binding corresponding to the appearance of
shifted bands 1a and 1b occurred shortly after a 450 cGy IR treatment;
in fact, slight increases were noted at t = 0, which was ~15 min
after exposure and analyses. In contrast, increases in TF binding
corresponding to shifted band 2 after 450 cGy were not apparent until
30 min, then dramatically increased over the next 90 min until peak
levels at 2 h (four- to fivefold increases) (Fig. 1A
)
were observed. Peak DNA binding activities for all TF complexes [i.e.,
Sp1 (band 1a) and the remaining RCPs (bands 1b and 2)] were observed
at 2 h (Fig. 1A
) after 450–600 cGy (Fig. 1B
). At higher doses of IR, induction of Sp1 binding was not
noted. A low level of nonspecific DNA binding comigrating with band 2
was observed, as evident when Sp1 cold competitor DNA was added (Fig. 1B
). Similar Sp1 binding patterns (i.e., formation of
shifted bands 1a, 1b, and 2, Figs. 1
and 2
) in log-phase nonirradiated
cells have been described (12
, 28)
. In addition, a minor
band (marked with an asterisk on Fig. 1B
) was sometimes
visible. The time and dose response of Sp1/RCP DNA binding induction
(Fig. 1A, B
) closely paralleled the induction of endogenous
c-fos, c-myc, c-jun, egr-1, and TK transcripts in confluent
U1-Mel cells (4
, 7)
. Induction of these IEGs (transcript
levels) was observed up to 600 cGy between 15 min and 4 h post
treatment, then rapidly declined both with time and with higher doses
of IR (4
, 7)
. Similarly, increases in Sp1/RCP DNA binding
were not observed after higher doses (over 750 cGy) of IR (Fig. 1B
). Increases in TFs binding to AP-1 consensus sites were
not observed.
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Increases in NF-B DNA binding activity were also observed
(Figs. 1C, D
). Unlike Sp1/RCP binding, increases in NF-B
binding were noted after 30 min and observed continuously for the next
24 h after 450 cGy. Peak DNA binding activities (two- to threefold
increases) were noted between 12 and 24 h (Fig. 1C
)
with 300–450 cGy (Fig. 1D
). After 48 h, NF-B
binding decreased to nonirradiated control levels. Aside from lowered
DNA binding at the 6 h time point due to loading, Oct-1 TF binding
was not altered by IR exposure. Increased NF-B DNA binding
corresponded in time to endogenous late IR-response gene expression in
U1-Mel cells, such as t-PA (4
, 15
, 16)
, but not to the
induction of IEGs. We previously observed that IR-inducible t-PA
expression correlated to NF-B binding within the promoter region of
t-PA gene by primer extension DNase I footprinting (16)
.
Together, these data suggest that t-PA may be controlled in part by
NF-B.
Alterations in p53 after IR have been well documented (29)
(reviewed in ref 13
). We examined the DNA binding activity
of p53 to its corresponding consensus site in confluent U1-Mel cells
before and after IR exposure. p53 DNA binding activity was greatly
increased 2 h after a high dose (1200 cGy) of IR treatment, but
increased only slightly at lower doses (450 cGy) (Fig. 1E
).
Peak p53 binding was consistently observed 2 h after high doses of
IR. Addition of antibodies that are directed against wild-type p53 in
band shift assays blocked p53-mediated DNA binding (Fig. 1E
). In contrast, mouse IgG had no effect on IR-activated
p53 DNA binding activity (data not shown).
Composition of Sp1/RCP complexes
We then investigated the composition of TFs binding to the
Sp1 site using monoclonal or polyclonal antibodies against Sp1 or pRb
(Fig. 2A
). Antibodies against Sp1 caused a supershift of band 1a
(see arrow in lanes 2, 5, and 8, Fig. 2A
) and weakened, but
did not eliminate, DNA binding at bands 1b and 2. These data suggest
that Sp1 (band 1a) may facilitate the binding of other RCPs (bands 1b
and 2) as previously proposed (12
, 28
, 30)
. Binding within
the supershifted band increased after IR treatment (compare lane 2 as
to nonirradiated nuclear extract with lanes 5 and 8, given 300 and 450
cGy, respectively) (Fig. 1)
. Addition of antibodies directed against
pRb (Fig. 2A
) had no effect on Sp1/RCE DNA binding (lanes 3,
6, and 9; Fig. 2A
), indicating that pRb was not a component
of the Sp1/RCP TF complexes observed in Fig. 1A, B
. These
data are consistent with previous reports that pRb expression affects
binding of Sp1 and RCPs to their respective consensus sequences, but
pRb itself is not a direct component of the DNA binding complex
(12
, 28
, 30)
.
Alterations in nuclear protein levels of Sp1, pRb, and p53 and
phosphorylation status of pRb and Sp1 In U1-Mel cells after IR
Immediate increases in the phosphorylated form of Sp1 (Sp1-P
band, Fig. 2B
) (18
, 31)
were noted in confluent
U1-Mel cells after IR, with peak levels appearing in 2 h. Scanning
densitometry revealed that nearly 20% of the total Sp1 protein was
phosphorylated (shifted upwards) in the nuclei of confluent U1-Mel
cells after doses of IR between 300 and 600 cGy. Higher doses of IR
(>750 cGy) resulted in very little phosphorylation of Sp1 (Fig. 2B
) compared to basal control levels, consistent with a lack
of Sp1 TF binding (Fig. 1B
). These data are consistent with
previous reports that phosphorylation of Sp1 increased its
transcriptional activity (31
, 32)
. The phosphorylation
status of pRb (Fig. 2C
) was not altered after IR, and
virtually all of the pRb protein present in the nucleus of quiescent
U1-Mel cells was in the hypophosphorylation form. These data suggest
that increases in Sp1 DNA binding in U1-Mel cells after IR may be
causally related to increased Sp1 phosphorylation (31
, 32)
and may regulate xips. Furthermore, coordinate increases in Sp1 and
NF-B DNA binding may regulate gene expression in a manner similar to
that proposed for HIV gene regulation (33)
.
We also examined alterations in the nuclear levels of p53 protein
in U1-Mel cells after IR (Fig. 2D
). U1-Mel cells express
wild-type p53 protein as determined by SSCP analyses (data not shown).
Nuclear p53 protein levels greatly increased in U1-Mel cells 2 h
after 1200 cGy, but only slight increases were noted after 450 cGy
(Fig. 2D
). Increases in nuclear p53 protein levels after
high doses of IR were consistent with increases in IR-activated p53 DNA
binding activity (Fig. 1E
), as described previously
(34
, 35)
.
Disruption of Sp1, NF-B, and p53 regulation result in various
cellular responses after IR
To further understand the roles of these TFs in IR responses, we
transfected U1-Mel cells with E6 (to block p53 responses) or E7 (to
block pRb regulation of Sp1) human papillomavirus genes under the
control of CMV promoters as described in Materials and Methods. An
estimated fivefold increase in basal Sp1/RCP DNA binding by
PhosphorImager analyses in E7-transfected U1-Mel (U1-Mel-E7) cells was
observed (Fig. 3A
). Sp1/RCP DNA binding remained unaltered in U1-Mel-E7 cells
after 5 or 10 Gy, in contrast to IR-activated binding previously
observed in parental U1-Mel or vector-alone transfected U1-Mel cells
(Fig. 1B
). Thus, loss of pRb function due to E7 transfection
resulted in an abrogation of IR-mediated Sp1/RCP DNA binding
activation. In a separate experiment using pRb-deficient human bladder
cancer cells (36)
, higher Sp1 DNA basal binding and no
induction after IR were also observed (C. R. Yang et al.,
unpublished data). U1-Mel-E7 cells showed loss of radioresistance
(i.e., loss of PLDR) compared to U1-Mel-vec1 (vector alone) or
U1-Mel-E6, as measured using colony-forming assays (Fig. 3B
). The D0 values were 220 cGy for
U1-Mel-vec1 or U1-Mel-E6 compared to 180 cGy for U1-Mel-E7. Not only
was the shoulder region of IR-treated, E7-transfected U1-Mel cells
statistically different (P<0.05 for 500 cGy) from E6- or
vector-alone transfected U1-Mel cells, but exponential IR-mediated cell
killing was also influenced. Loss of survival mediated by IR under
these confluent conditions presumably was due to dramatically increased
apoptosis in IR-treated U1-Mel-E7 cells compared to U1-Mel-vec1 or
U1-Mel-E6 cells (Table 1
); apoptotic cell populations were determined using flow cytometry,
Hoescht dye staining, and by monitoring changes in morphology as
described in Materials and Methods. The plating efficiency of U1-Mel-E6
cells was dramatically reduced (3% vs. 50% in U1-Mel-vec1 or -E7).
However, those cells that did attach retained E6 expression and were
not altered in their sensitivity to IR compared to U1-Mel-vec1 cells
(Fig. 3B
). In contrast, U1-Mel cells overexpressing the
IB S32/36A mutant protein (U1-Mel-IB S32/36A, which blocks
nuclear translocation of NF-B (37)
, showed enhanced
radioresistance (as measured by PLDR) compared to U1-Mel-vec2 (vector
alone) cells (Fig. 3C
).
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Both IR-treated U1-Mel-E6 and U1-Mel-E7 cells demonstrated a lack of
G0/G1 cell cycle checkpoint
arrest, a concomitantly enhanced accumulation of irradiated cells in
G2/M, and increased population of polyploidy
cells compared to U1-Mel-vec1 cells after IR (Table 1)
. No obvious cell
cycle checkpoint differences or increases in apoptosis or polyploid
cell populations were observed in U1-Mel-IB S32/36A cells
compared to U1-Mel-vec2 (vector alone) cells after IR. A similar
increase in polyploid cell populations was demonstrated in
p21-deficient colon cancer cells after IR (38)
. These data
strongly suggest that the consequence of deregulation of pRb, Sp1, or
p53 to the surviving cells after IR appears to be a dramatic loss of
cell cycle checkpoint responses and a concomitant dramatic increase in
genomic instability, as measured by the emergence of polyploid U1-Mel
cells (Table 1)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and high rates of PLDR after IR
(3)
. For example, after 2–10 Gy, confluent U1-Mel cells
demonstrated survival increases of three- to fivefold, respectively, in
2–8 h if held in a quiescent state before replating for colony-forming
ability. The experiments performed here were to better understand the
transcriptional responses of these cells during DNA repair and survival
recovery responses (i.e., PLDR), since we previously described the
X-ray induction of endogenous TK, IEGs, and t-PA (4
, 6
, 7
, 15)
transcripts and proteins in U1-Mel cells. Our previous data
suggest that IR induces only a certain subset of genes (including many
of the IEGs), resulting in the stimulation of transcripts/proteins
perhaps needed for DNA repair or cell cycle checkpoints
(7)
.
Activation of NF-B DNA binding appears to occur later than Sp1/RCP
DNA binding after IR (Fig. 1)
. However, activation of both TFs
demonstrated similar dose response kinetics, being induced at doses up
to 600 cGy but declining with higher doses of IR. NF-B and Sp1 peak
DNA binding affinities/levels occurred at different times after IR
treatment (Fig. 1)
. Although NF-B DNA binding activity appeared
within the first 1–2 h after IR, peak DNA binding activity and
expression of the NF-B-responsive, t-PA transcript were apparent
10–12 h after IR (15)
. Both NF-B and Sp1/RCP DNA
binding as well as IEG, t-PA, TK, and certain xip expression
demonstrated inducible activity at low levels of IR, but were
down-regulated after high doses (>600 cGy) (Fig. 1)
(7)
.
The significance of the coordinate activation of Sp1/RCP and NF-B
DNA binding after IR is not known nor is the mechanism of reduced TF
binding and subsequent reduced gene expression after high doses of IR.
Sp1/RCP and NF-B proteins may synergistically interact on certain
promoter regions (33
, 39
40
41)
. These data suggest that
these two TFs may be activated coordinately after IR and work
cooperatively to control gene transcription in response to DNA damage
in certain cells.
The IR-activated DNA binding of Sp1/RCP to Sp1 consensus sites
was very complex, resulting in three mobility-shifted DNA–protein
complexes appearing as bands 1a, 1b, and 2. Only band 1a contained the
Sp1 protein (Fig. 1A
), and DNA binding of the three bands
increased at different rates after IR. Recently, RCP genes with
conserved Sp1 DNA binding domain have been discovered (e.g., Sp2, Sp3,
Sp4) (42)
. Understanding the sequence of Sp1 and RCP
binding to Sp1/RCE sites in certain responsive promoters after IR will
be central to our understanding of the mechanisms and complexities
behind IR-inducible gene transcription. It is interesting that we found
no increases in AP-1 DNA binding activity in U1-Mel cells after IR
(5
, 6)
. We expected increases in this transcription
factor, since other laboratories have described increases in
c-jun/c-fos heterodimeric DNA binding after IR in other cell systems
(43
, 44)
. Our data indicate that at clinically relevant
doses of IR (200 cGy), IEG induction may occur by AP1-independent but
Sp1/RCP-dependent mechanisms in radioresistant human U1-Mel melanoma
cells. We propose that such induction occurs through Sp1/RCP in
combination with NF-B. Further research is required to test this
hypothesis, and will be essential before one could logically use
IR-responsive promoters (XRE) for gene therapy (16
, 45)
.
How does Sp1/RCP and NF-B activation of DNA binding occur after IR?
Sp1 and IB (NF-B inhibitor) are substrates for DNA-dependent
protein kinase (DNA-PK) in vitro (18
, 19
, 21)
.
This kinase (DNA-PKcs, catalytic subunit) requires free DNA ends and
associates with the Ku antigen (46)
, an heterodimer of p70
(Ku70) and p80 (Ku80) protein subunits. DNA-PK is intimately involved
in DNA double-strand break repair (47
, 48)
.
Phosphorylation of Sp1 increases its transcriptional activity
(31
, 32)
, whereas phosphorylation and subsequent
proteolysis of IB release and activate NF-B (37)
.
p53 is also a substrate for DNA-PK in vitro, and it is
feasible that phosphorylation of p53 at Ser15 by DNA-PK may stabilize
p53 and increase its half-life in the nucleus (49)
.
Previously published data proposed that DNA-PK acts upstream of p53 in
response to DNA damage (20)
. We hypothesize that DNA-PK is
a DNA damage checkpoint modulator (7)
. It recognizes and
participates in the repair of single- and double-strand DNA breaks
generated by IR. In response, DNA-PK may transduce the signal of DNA
damage through phosphorylation, perhaps coordinately activating Sp1,
NF-B (via IB phosphorylation), or p53. As our data in Figs. 1
and 2
indicate, the levels of these TFs in the nucleus appear to be
coordinately regulated; data suggest that the extent of damage
initially created may determine the spectrum of responses observed. Low
doses of IR would activate high-affinity substrates, such as Sp1,
whereas relatively higher doses of IR would be required to activate
sufficient DNA-PKcs enzyme activity to phosphorylate low-affinity
substrates such as p53. The affinities may be altered by the growth
status of the cell. Confluent U1-Mel cells were used exclusively in our
system.
We speculate that DNA-PK phosphorylates Sp1 and possibly other
RCPs in vivo, resulting in a cellular decision to carry out
DNA repair after low doses (clinically relevant) of IR. It has been
shown that both Ku80 and DNA-PKcs gene expression are Sp1 dependent
(50
, 51)
. In contrast, high doses of IR create badly
damaged cells with large amounts of DNA breaks resulting in peak DNA-PK
activation, leading to phosphorylation and stabilization of p53, and
thereby increased p53 nuclear levels (Fig. 3)
(52
, 53)
.
Sp1/RCP binding rapidly decreased with high doses of IR. This may
actually be regulated by p53 expression (54)
. We speculate
that the inverse relationship between Sp1/RCP and p53 binding may
control a cellular decision to undergo DNA repair or p53-dependent
apoptosis (28
, 55)
. Other candidates for this upstream
modulator are the ATM (ataxia telangiectasia mutation) or c-abl
kinases. Emerging data suggest that the ATM kinase phosphorylates and
regulates p53 (56
, 57)
and IB (14
, 58)
.
Functional knockout experiments (Fig. 3
and Table 1
) further suggest
that certain TFs may be directly or indirectly involved in cell
survival or cell death. Disruption of pRb (E7 transfection), and
therefore Sp1 regulation, resulted in loss of the radioresistant
phenotype of U1-Mel cells (Fig. 3B
); the significance of Sp1
vs. other factors regulated by pRb cannot be distinguished at this
time. However, the data presented here clearly indicate that pRb is a
major factor in protecting cells from apoptosis, as well as from the
generation of polyploid cell populations (Table 1)
.
In contrast, deregulation of NF-B by the IB S32/36A
dominant-negative mutant protein enhanced the radioresistance of U1-Mel
cells (Fig. 3C
). These data appear contrary to previous
studies showing an anti-apoptotic role for NF-B after tumor necrosis
factor exposure (26
, 59)
.
Similar increases in polyploid U1-Mel-E6 and U1-Mel-E7 cell populations
at similar time frames after IR exposure were noted (Table 1)
. These
data highlight the importance and consequences of cell cycle checkpoint
abrogation. Using these model systems, we hope to better understand the
role(s) of IR-inducible TFs and genes in cell cycle checkpoints,
radiosensitivity, PLDR, apoptosis and in the maintenance of genomic
stability, which will be clinically important to sensitize
radioresistant tumors for radiotherapy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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