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J Biol Chem, Vol. 273, Issue 21, 13030-13036, May 22, 1998
From the Program in Gene Regulation, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912
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ABSTRACT |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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The ability of p53 to induce apoptosis requires
its sequence-specific DNA binding activity; however, the
transactivation-deficient p53(Gln22-Ser23) can still induce
apoptosis. Previously, we have shown that the region between residues
23 and 97 in p53 is necessary for such activity. In an effort to more
precisely map a domain necessary for apoptosis within the N terminus,
we found that deletion of the N-terminal 23 amino acids compromises,
but does not abolish, p53 induction of apoptosis. Surprisingly,
p53(
1-42), which lacks the N-terminal 42 amino acids and the
previously defined activation domain, retains the ability to induce
apoptosis to an even higher level than wild-type p53. A more extensive
deletion, which eliminates the N-terminal 63 amino acids, renders p53
completely inert in mediating apoptosis. In addition, we found that
both p53(1-42) and p53(Gln22-Ser23) can
activate a subset of cellular p53 targets. Furthermore, we showed that
residues 53 and 54 are critical for the apoptotic and transcriptional
activities of both p53(1-42) and
p53(Gln22-Ser23). Taken together, these data
suggest that within residues 43-63 lie an apoptotic domain as well as
another transcriptional activation domain. We therefore postulate that
the apoptotic activity in p53(Gln22-Ser23) and
p53(1-42) is still transcription-dependent.
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INTRODUCTION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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The p53 tumor suppressor protein serves as a checkpoint in maintaining genome stability (1-3). Several different biological responses that could play a role in maintaining genome stability have been strongly correlated with wild-type p53 function (1, 3). Following stress conditions such as in the presence of damaged DNA or insufficient growth and survival factors, the cellular levels of p53 increase. This leads to one of at least three well understood cellular responses as follows: cell cycle arrest, differentiation, or apoptosis. Several factors have been shown to determine how a cell responds to the accumulation of p53, e.g. cell type and the presence of several cellular and viral proteins (4-8). In addition, the levels of p53 in a given cell can dictate the response of the cell such that lower levels of p53 result in cell cycle arrest (9) or differentiation (10), whereas higher levels result in apoptosis (9, 10).
The functional domains of p53 have been subjected to extensive analysis (1, 3, 4). A transcriptional activation domain has been shown to lie within N-terminal residues 1-42 (11, 12). Within this region there are a number of acidic and hydrophobic residues, characteristics of the acidic activator family of transcriptional factors (13). Indeed, a double point mutation of the two hydrophobic amino acids at residues 22 and 23 renders p53 transcriptionally inactive (14). These two residues presumably are required for the interaction of the activation domain with the TATA box binding protein and/or TATA box binding protein-associated factors (15-18).
It is well established that as a transcriptional activator, p53 up-regulates p21, a cyclin-dependent kinase inhibitor (19-21), which leads to p53-dependent G1 arrest. However, it is not certain what function(s) of p53 is required for apoptosis. The transactivation function of p53 was shown to be required in some experimental protocols (22-24). There are several candidate genes that play roles in apoptosis that can be activated in response to p53 induction, such as BAX (25), IGFBP3 (26), PAG608 (27), KILLER/DR5 (28), and several redox-related PIGs genes (29). Several other studies, including our own observations, have provided evidence that p53 might have a transcription-independent function in apoptosis (9, 30-32). Recently, the proline-rich region between residues 60 and 90, which comprises five "PXXP" motifs (where P represents proline and X any amino acid), was found to be necessary for efficient growth suppression (33) and apoptosis (34) and to serve as a docking site for transactivation-independent growth arrest induced by GAS1 (35).
Previously, we showed that the region between residues 23 and 97 is necessary for apoptosis (9). To more precisely map such a domain in the N terminus necessary for apoptosis, we have made several new mutants. Analyses of these mutants lead to identification of a novel domain between residues 43 and 63 that can mediate apoptosis and activate cellular p53 targets. We also found that a double point mutation at residues 53 and 54 completely abolishes both the transcriptional and apoptotic activities mediated by this novel domain. Thus, we hypothesize that a transcriptional activity located in this novel domain regulates a subset of cellular p53 targets that are responsible for apoptosis.
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EXPERIMENTAL PROCEDURES |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Plasmids and Mutagenesis--
Mutant p53 cDNAs were
generated by polymerase chain reaction using the full-length wild-type
p53 cDNA as a template. To generate p53(1-23), the pair of
primers used were as follows: forward primer N24, GAT CGA ATT CAC CAT
GGG CTA CCC ATA CGA TGT TCC AGA TTA CGC TAA ACT ACT TCC TGA A; and
reverse primer C393, GAT CGA ATT CTC AGT CTG AGT CAG GCC CTT. To
generate p53(1-42), the pair of primers used were as follows:
forward primer N43, GAT CGA ATT CAC CAT GGG CTA CCC ATA CGA TGT TCC AGA
TTA CGC TTT GAT GCT GTC CCC G; and reverse primer C393. To generate
p53(1-63), the pair of primers used were: forward primer N64, GAT
CGA ATT CAC CAT GGG CTA CCC ATA CGA TGT TCC AGA TTA CGC TCC CAG AAT GCC
AGA GGC T; and reverse primer C393. To generate
p53(Gln22-Ser23/Gln53-Ser54),
cDNA fragments encoding amino acids 1-59 and 60-393 were
amplified independently and ligated together through an internal
AvaII site. The p53(Gln22-Ser23)
cDNA was used as a template (14). The pair of primers for the
cDNA fragment encoding amino acids 1-59 were as follows: forward primer N1, GAT CGA ATT CAC CAT GGG CTA CCC ATA CGA TGT TCC AGA TTA CGC
TGA GGA GCC GCA GTC AGA TCC; and reverse primer C59, TTC ATC TGG ACC
TGG GTC TTC AGT GCT CTG TTG TTC AAT ATC. The pair of primers for the
cDNA fragment encoding amino acids 60-393 were as follows: forward
primer N60, ACT GAA GAC CCA GGT CCA; and reverse primer C393. To
generate p53(1-42/Gln53-Ser54), the
p53(Gln22-Ser23/Gln53-Ser54)
cDNA was amplified by forward primer N43 and reverse primer C393.
Mutations were confirmed by DNA sequencing.
Cell Lines, Transfection, and Selection Procedures--
The
H1299 cell line was purchased from the American Type Culture Collection
and grown with Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum at 37 °C with 5% CO2. Transfections were performed using the calcium chloride method as
described (36). Cell lines expressing inducible proteins of interest
were generated as described previously (9). Individual clones were
screened for inducible expression of the p53 protein by Western blot
analysis using monoclonal antibodies against p53. The H1299 cell lines
that inducibly express either wild-type p53 or p53(364-393) are
p53-3 and p53(364-393)-1, respectively, as described previously
(9). The H1299 cell line that inducibly expresses p53(62-91) is
p53(62-91)-5.1
Western Blot Analysis-- Cells were collected from plates in phosphate-buffered saline, resuspended with 1× sample buffer, and boiled for 5 min. Western blot analysis was performed as described previously (37). Monoclonal antibodies used to detect p53 were Pab240 and Pab421 (37). The affinity purified monoclonal antibody against p21 (Ab-1) was purchased from Oncogene Science (Uniondale, NY). Affinity purified anti-actin polyclonal antibodies were purchased from Sigma.
Growth Rate Analysis-- To determine the rate of cell growth, cells were seeded at 5-10 × 104 cells per 60-mm plate, with or without tetracycline (2 µg per ml). The medium was replaced every 72 h. At times indicated, two plates were rinsed with phosphate-buffered saline twice to remove dead cells and debris. Live cells on the plates were trypsinized and collected separately. Cells from each plate were counted three times by Coulter cell counter. The average number of cells from at least two plates were used for growth rate determination.
FACS2 Analysis-- Cells were seeded at 2.0 × 105 per 90-mm plate with or without tetracycline. Three days after plating, both floating dead cells in the medium and live cells on the plate were collected and fixed with 2 ml of 70% ethanol for at least 30 min. For FACS analysis, the fixed cells were centrifuged and resuspended in 1 ml of phosphate-buffered saline solution containing 50 µg/ml each of RNase A (Sigma) and propidium iodide (Sigma). The stained cells were analyzed in a fluorescence-activated cell sorter (FACSCaliber, Becton Dickinson) within 4 h. The percentage of cells in sub-G1, G0-G1, S, and G2-M phases was determined using the ModFit program. The percentage of cells in sub-G1 phase was used as an index for the degree of apoptosis.
Cell Viability Assay by Trypan Blue Exclusion-- Cells were seeded at 2 × 105 per 90-mm plate with or without tetracycline. Three days after plating, both floating cells in the medium and live cells on the plate were collected and concentrated by centrifugation. After stained with trypan blue (Sigma) for 15 min, both live (unstained) and dead (stained) cells were counted two times in a hemocytometer. The percentage of dead cells from control plates was subtracted from the percentage of dead cells from experimental plates, and the resulting value was used as an index for the degree of apoptosis.
RNA Isolation and Northern Blot Analysis-- Total RNA was isolated using Trizol reagent (Life Technologies, Inc.). Northern blot analysis was performed as described (37). The p21 probe was made from an 1.0-kilobase pair EcoRI-EcoRI fragment (19); the MDM-2 probe was made from a 2.1-kilobase pair NotI-SmaI fragment (38); the BAX probe was made from a 290-base pair PstI-BglII fragment (39); the GADD45 probe was from a 400-base pair EcoRI-BamHI fragment (40); the GAPDH probe was made from an 1.25-kilobase pair PstI-PstI cDNA fragment (41); and the MCG14 cDNA probe was a 200-base pair polymerase chain reaction fragment identified by CLONTECH PCR-Select cDNA subtraction.2
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RESULTS |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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A Novel Domain within Residues 43-63 Is Necessary for Mediating
Apoptosis--
Previously, we showed that p53(1-22), which lacks
the N-terminal 22 amino acids, can still induce apoptosis as well as
cell cycle arrest (9). Since both residues 22 and 23 are critical for
p53 transcriptional activity (14), we decided to determine whether
p53(1-23), which deletes the N-terminal 23 amino acids, would also
be able to induce apoptosis and activate cellular p53 targets.
1-23). Three representative cell lines,
p53(1-23)-9, -10, and -23, are shown in Fig.
1A. Western blot analysis
showed that these cell lines express p53(1-23) at levels comparable
to wild-type p53 in p53-3 cells (Fig. 1A). To characterize
p53(1-23), we looked at its transcriptional and apoptotic
activities and the growth rate of the cell line p53(1-23)-9. The
transcriptional activity was determined by monitoring the expression of
the endogenous gene, p21, a well defined transcriptional target of p53
(19). We found that p53(1-23) is still capable of activating p21,
albeit to a much less degree than wild-type p53 (Fig. 1A).
Next, the growth rates of p53(1-23)-9 cells under both uninduced
and induced conditions were determined, and these cells failed to
multiply following p53 expression (Fig. 1B). To exclude
potential effects of the regulator tetracycline and/or the tet-vp16
transactivator (42) on cell growth, we analyzed the growth rate of the
cell line H24-1, which was similarly established but did not express
any protein. The results showed that the growth rates of H24-1 cells
under both the uninduced (+tet) and induced (
tet) conditions were
nearly identical (Fig. 1C), indicating that both
tetracycline and tet-vp16 transactivator have no effect on cell growth.
It is well established that the percentage of cells containing a
sub-G1 DNA content reflects the extent to which cells are
undergoing apoptosis (9, 23, 31). Since p53 can induce apoptosis in
H1299 cells (9, 31), FACS analysis was used to observe the extent of
apoptosis by determining the distribution of cells in each phase of the
cell cycle. The results showed that 18% of cells expressing
p53(1-23) had a sub-G1 DNA content 3 days after
induction of this mutant, compared with less than 5% of the same cells
expressing no p53 (Fig. 1, D and E; Table
I). Trypan blue exclusion assay showed
that 15% of cells were dead, which is consistent with FACS analysis.
In contrast, about 45 and 30% of cells had a sub-G1 DNA
content at day 3 following expression of either wild-type p53 or
transactivation-deficient p53(Gln22-Ser23),
respectively (Table I). The FACS results also showed that the number of
cells in S phase was decreased from 38 to 22.3% following induction of
p53(1-23), and these cells primarily arrested in G1
(Fig. 1, D and E). Similar results were obtained
using another high p53(1-23) producer, p53(1-23)-10.
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1-23) is still capable of inducing apoptosis and p53
activation domain lies within residues 1-42 (11, 12), we determined
whether the other half (residues 24-42) of the previously defined
activation domain is required for apoptosis. To this end, we
established 16 individual stable cell lines that inducibly express
p53(1-42) that lacks the N-terminal 42 amino acids. Three representative cell lines, p53(1-42)-2, -5, and -11, were shown in
Fig. 2A. Consistent with
previous results that p53(Gln22-Ser23) cannot
activate p21 (9, 22, 33, 43), p53(1-42) only minimally activated
p21 as compared with wild-type p53 (Fig. 2A). We then
determined the growth rate of a high producer, p53(1-42)-2. Surprisingly, we found that a majority of cells died within 3 days
following induction of p53(1-42) (Fig. 2B). In addition, both trypan blue exclusion assay and FACS analysis showed that approximately 50-68% of cells underwent apoptosis (Fig.
2C; Table I). Similar results were obtained from several
other cell lines. These results suggest that the entire previously
defined activation domain within the N-terminal 42 amino acids is
dispensable for apoptosis. In fact, deletion of this region enhanced
the ability of p53 to induce apoptosis (Table I).
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1-63) which lacks the N-terminal 63 amino acids but contains an
intact proline-rich region. Three representative cell lines, p53(1-63)-14, -22, and -27, were shown in Fig.
3A, and the activity of
p53(1-63) was analyzed as above. The results showed that
p53(1-63) was unable to activate p21 expression (Fig.
3A), and p53(1-63)-14 cells, a high p53 producer,
continued to multiply when p53(1-63) was induced (Fig.
3B). Furthermore, both FACS analysis and trypan blue
exclusion assay showed that neither apoptosis nor cell cycle arrest was
observed in cells expressing p53(1-63) (Fig. 3C and Table I).
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Within Residues 43-63 Lies Another Activation Domain That Overlaps
with the Domain Necessary for Mediating Apoptosis--
The ability of
transactivation-deficient p53(Gln22-Ser23) to
induce apoptosis leads to the hypothesis that p53 has
transcription-independent apoptotic activity (9, 31, 33). Since
p53(1-42) lacks the previously defined activation domain and only
minimally activates p21 as determined by Western blot analysis (Fig.
2A), it appears that it can induce apoptosis in a
transcription-independent manner. To ascertain whether p53(1-42)
contains a transcriptional activity, the expression patterns of four
well defined cellular p53 targets, p21, MDM2,
GADD45, and BAX, were analyzed in cells
expressing p53(1-42) by Northern blot analysis (Fig.
4A). The expression levels of
these genes in cells with or without p53 were quantitated by
PhosphorImage scanner, and the fold increase of their relative mRNAs was calculated after normalization to
glyceraldehyde-3-phosphate dehydrogenase mRNA levels (Table
II). The results showed clearly that
p53(1-42) significantly activated MDM2 (8-fold),
GADD45 (7.03-fold), and BAX (3.9-fold) but only
minimally activated p21 (1.83-fold). As expected, wild-type
p53 but not mutant p53(Gln22-Ser23) activated
these cellular p53 targets (Fig. 4A; Table II). As a
control, p53(64-91), which lacks all of the five PXXP
motifs, was examined. The proline-rich domain in p53 is dispensable for transactivation (33, 34). As expected, p53(64-91) activated these
p53 targets (Fig. 4A and Table II). Since p53(1-63)
failed to activate any of these p53-regulated genes (data not shown), the results suggest that another activation domain lies within residues
43-63. For clarity, we designate the originally defined activation
domain located within residues 1-42 as activation domain I and this
novel domain as activation domain II.
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1-42), p53(64-91), and p53(364-393)
(Fig. 4B).
Since a double point mutation at residues 22 and 23 abolishes the
transcriptional activity of the activation domain I (14), we looked for
analogous hydrophobic amino acids within the activation domain II. Two
were found: tryptophan at residue 53 and phenylalanine at residue 54. We therefore made identical mutations in these two amino acids in
p53(Gln22-Ser23) or p53(1-42), changing
tryptophan 53 to glutamine and phenylalanine 54 to serine to generate
p53(Gln22-Ser23/Gln53-Ser54)
and p53(1-42/Gln53-Ser54). We then
established a number of cell lines that inducibly express these
mutants, and their ability to induce apoptosis and activate cellular
p53 targets were similarly analyzed as above. Three representative cell
lines that express either
p53(Gln22-Ser23/Gln53-Ser54)
or p53(1-42/Gln53-Ser54) are shown in Fig.
5, A and C,
respectively. As expected, Western blot analysis showed that p21 was
not activated by either of these mutants (Fig. 5, A and
C, bottom panel). In addition, these mutants were unable to
induce apoptosis, as demonstrated by the rate of cell growth (Fig. 5,
B and D), trypan blue exclusion assay, and FACS
analysis (Table I). Furthermore, the putative cellular p53 target
MCG14, which can be activated by p53(1-42) and
p53(Gln22-Ser23) (Fig. 4B), failed
to be activated in cells expressing either p53(Gln22-Ser23/Gln53-Ser54)
or p53(1-42/Gln53-Ser54) (Fig.
4C). These results indicate that residues 53 and 54 are critical for the novel domain within residues 43-63 to induce apoptosis and activate cellular p53 targets.
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DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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The p53 protein has been divided into several functional domains (1, 3, 4) as follows: (a) an activation domain which lies within residues 1-42 that has been shown to be required for both transcriptional activation and repression (11, 12, 14, 44); (b) a newly identified proline-rich domain within residues 64-91 which is necessary for efficient growth suppression (33), apoptosis (34), and for mediating GAS1-dependent growth arrest (35); (c) a sequence-specific DNA-binding domain which lies within the central, conserved portion of the protein (1, 3); (d) a nuclear localization signal which lies within residues 316-325 (1, 3); (e) a tetramerization domain which lies within residues 334-356 (1, 3); and (f) a C-terminal basic domain which binds DNA nonspecifically and regulates the sequence-specific DNA binding activity (1, 3).
Here we found that within residues 43-63 lies another novel domain
that is necessary for apoptosis on the basis of the following observations: (i) p53(1-42), which lacks the N-terminal 42 amino acids and the previously defined activation domain, contains a strong
apoptotic activity; (ii) p53(1-63), which lacks the N-terminal 63 amino acids but contains intact PXXP motifs, has no
apoptotic activity; (iii) a double point mutation at residues 53 and 54 renders both p53(1-42/Gln53-Ser54) and
p53(Gln22-Ser23/Gln53-Ser54)
completely inert in inducing apoptosis; and (iv) codon 53 is one of the
frequently mutated sites outside the DNA binding domain in the
p53 gene in human tumors (45), which underscores the importance of the apoptotic function within residues 43-63 in p53
tumor suppression.
How does this novel domain mediate an apoptotic activity? Previously,
it was shown that p53(Gln22-Ser23), which
cannot activate several cellular p53 targets (9, 14, 22, 31, 43), is
still capable of inducing apoptosis (9, 31, 34), and a p53 mutant,
which lacks the proline-rich region, is capable of activating several
p53 targets (33, 34) but cannot induce apoptosis (33, 34). These
results lead to a hypothesis that p53 has both
transcription-dependent and -independent functions in
apoptosis. However, it is well established that p53 mutants that are
defective in sequence-specific DNA binding activity are also inert in
inducing apoptosis (1, 3, 4), suggesting that p53 sequence-specific DNA
binding activity and possibly its sequence-specific transcriptional
activity are required for inducing apoptosis. Here we found that
p53(1-42), which lacks the entire previously defined activation
domain I, not only induces apoptosis, but also activates the
MDM2, BAX, and GADD45 genes through
its activation domain II located between residues 43 and 63 (Fig. 4;
Table II). Since p53(Gln22-Ser23) contains an
intact activation domain II, we hypothesized that it might still
contain transcriptional activity. Indeed, we found that
p53(Gln22-Ser23) can activate one of the
putative p53 targets, MCG14. Furthermore, a double point
mutation at residues 53 and 54 completely abolishes the ability of both
p53(Gln22-Ser23/Gln53-Ser54)
and p53(1-42/Gln53-Ser54) to activate MCG14
and induce apoptosis. Consistent with our results, Candau et
al. (46) recently showed that within residues 40-83 lies a
sub-activation domain, which can activate a reporter gene under control
of a promoter with a p53-responsive element when p53 is cotransfected,
and a double point mutation at residues 53 and 54 also abolished the
transcriptional activity of the sub-activation domain. These results
suggest that p53 has two independent activation domains. A second
activation domain within a transcription factor is not without
precedent. Herpes simplex virus protein VP16 also contains two
independent activation domains (47). Thus, it appears that in response
to various stress conditions and their subsequent modifications, the
two independent activation domains might serve as an intrinsic factor
of p53 that determines whether a given p53 target is activated.
Although BAX, MDM2, and GADD45 are the activation domain II-regulated gene products, these cellular p53 targets might not mediate the p53-dependent apoptosis on
the basis of two observations: (i) these genes were not activated by
p53(Gln22-Ser23) which is competent in inducing
apoptosis (Fig. 4A; Table II); (ii) these genes were
activated by p53(62-91) which is defective in inducing apoptosis
(Fig. 4A; Table II). Since cell type has been shown to
influence the cellular response (cell cycle arrest or apoptosis) to p53
(1, 4, 8), cellular genetic background might then determine the
modification of the two activation domains. Therefore, the results
obtained in H1299 cells need to be confirmed in other cell types.
It is intriguing that although p53(Gln22-Ser23)
contains an intact activation domain II, it fails to activate
BAX, GADD45, and MDM2 (Fig.
4A). Since both p53(1-23) and p53(1-42) can activate these p53 targets, it suggests that the presence of the first 23 amino
acids may mask the ability of the activation domain II in
p53(Gln22-Ser23) to activate these cellular p53
targets. Alternatively, it is also possible that when the activation
domain I is inactivated by a double point mutation at residues 22 and
23, the N-terminal 42 residues might then inhibit or block interaction
of a co-activator (or an adaptor) with the activation domain II that is
required for activation of some p53 targets, such as MDM2,
p21, BAX, and GADD45, but not for
activation of other p53 targets, such as MCG14. It is
important to note that although the activation domain I is primarily
responsible for activation of p21, the level of p21 in cells expressing
either p53(1-23) and p53(1-42) was slightly increased upon p53
induction (Figs. 1A and 2A), suggesting that the
activation domain II can weakly activate p21. Furthermore, our
preliminary studies showed that activation of p21 was compromised by a
double point mutation at residues 53 and 54 when
p53(Gln53-Ser54) was expressed at a low to
intermediate level,3
consistent with the idea that the activation domain II contributes to
the activation of p21. Since several clones that express various expression levels of the target genes are required for determining the
function of the targets (48), these results remain to be confirmed.
Previously, it was shown that overexpression of p21 can protect human
colorectal carcinoma RKO cells from prostaglandin A2-mediated apoptosis
(49). Lack of p21 expression due to homologous deletion of the
p21 gene also renders HCT116 colorectal cancer cells
susceptible to apoptosis following treatment with either 
-radiation
or chemotherapeutic agents (50). In addition, a significant fraction of
tumors in mice deriving from p21
/ HCT116 cancer cells
were completely cured, and all tumors deriving from p21+/+
cancer cells underwent regrowth after treatment with -radiation (50). It is interesting to note that
p53(Gln22-Ser23) and p53(1-42), both of
which lack a functional activation domain I, cannot significantly
activate p21 (Fig. 2 and 4; Table II) but can induce apoptosis (Table
I). The strong apoptotic activity conferred by p53(1-42) might be
due to its failure of activating p21. Thus, we have generated a mutant,
p53(1-42), that might be better than wild-type p53 in the
elimination of cancer cells and therefore a potential candidate for
gene therapy.
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ACKNOWLEDGEMENTS |
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We thank Bill Dynan and Howard Rasmussen for the initial setup of our core facility which makes this work possible and C. Prives for constant encouragement. We are grateful to Mark Anderson, Rhea Markowitz, and Dan Dransfield for their critical reading of this manuscript and many suggestions.
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FOOTNOTES |
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* This work was supported in part by Grants DAMD17-94-J-4142 and DAMD17-97-I-7019 from the United States Department of Army Breast Cancer Program and Grant CA76069-01 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: CB-2803/IMMAG, 1120 15th St., Medical College of Georgia, Augusta, GA 30912. Tel.: 706-721-8760; Fax: 706-721-8752; E-mail: xchen{at}mail.mcg.edu.
1 J. Zhu, W. Zhou, J. Jiang, and X. Chen, manuscript in preparation.
2 The abbreviation used is: FACS, fluorescence-activated cell sorter.
3 J. Zhu, W. Zhou, J. Jiang, and X. Chen,unpublished results.
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REFERENCES |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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A. Scoumanne and X. Chen The Lysine-specific Demethylase 1 Is Required for Cell Proliferation in Both p53-dependent and -independent Manners J. Biol. Chem., May 25, 2007; 282(21): 15471 - 15475. [Abstract] [Full Text] [PDF]
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D. P. Teufel, S. M. Freund, M. Bycroft, and A. R. Fersht Four domains of p300 each bind tightly to a sequence spanning both transactivation subdomains of p53 PNAS, April 24, 2007; 104(17): 7009 - 7014. [Abstract] [Full Text] [PDF]
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K. L. Harms and X. Chen Histone Deacetylase 2 Modulates p53 Transcriptional Activities through Regulation of p53-DNA Binding Activity Cancer Res., April 1, 2007; 67(7): 3145 - 3152. [Abstract] [Full Text] [PDF]
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Y. Xu and X. Chen Glyoxalase II, a Detoxifying Enzyme of Glycolysis Byproduct Methylglyoxal and a Target of p63 and p73, Is a Pro-survival Factor of the p53 Family J. Biol. Chem., September 8, 2006; 281(36): 26702 - 26713. [Abstract] [Full Text] [PDF]
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A. Scoumanne and X. Chen The Epithelial Cell Transforming Sequence 2, a Guanine Nucleotide Exchange Factor for Rho GTPases, Is Repressed by p53 via Protein Methyltransferases and Is Required for G1-S Transition. Cancer Res., June 15, 2006; 66(12): 6271 - 6279. [Abstract] [Full Text] [PDF]
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E. M. Hammond, D. J. Mandell, A. Salim, A. J. Krieg, T. M. Johnson, H. A. Shirazi, L. D. Attardi, and A. J. Giaccia Genome-Wide Analysis of p53 under Hypoxic Conditions. Mol. Cell. Biol., May 1, 2006; 26(9): 3492 - 3504. [Abstract] [Full Text] [PDF]
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) or absence (
) of p53 were measured as
described under "Experimental Procedures." C, growth
rates of H24-- cells in the presence ([
) of
tetracycline. D, DNA contents were quantitated by propidium
iodide staining of fixed cells at day 3 following withdrawal of
tetracycline as described under "Experimental Procedures."
E, The percentages of p53(
