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The Wistar Institute [K. S., N. H. C., M. J. F. W., M. C. L., M. H., T. D. H.], and Departments of Genetics and Medicine, Howard Hughes Medical Institute, University of Pennsylvania [W. S. E.], Philadelphia, Pennsylvania 19104-4268
Abstract
Sporadic human tumors and the hereditary cancer predisposition syndrome Li-Fraumeni are frequently associated with mutations in the p53 tumor suppressor gene that compromise its ability to function as a DNA damage checkpoint. A subset of Li-Fraumeni patients with wild-type p53 alleles have mutations in chk2/hcds1, one of the genes signaling the presence of DNA damage to the p53 protein. This suggests that p53 may be kept inactive in human cancer by mutations targeting DNA damage signaling pathways. Melanoma cells are highly radioresistant, yet they express wild-type p53 protein, raising the possibility of defects in the pathways that activate p53 in response to DNA damage. We have described a chk2/hcds1-independent DNA damage signaling pathway that targets Ser-376 within the COOH terminus of p53 for dephosphorylation and leads to increased p53 functional activity. We now report that in several human melanoma cell lines that express wild-type p53, the phosphorylation state of Ser-376 was not regulated by DNA damage. In these cell lines, neither the endogenous wild-type p53 protein nor high levels of ectopic wild-type p53 led to cell cycle arrest or apoptosis. Thus, defective activation of p53 in response to DNA damage may underlie the radioresistance of human melanoma cells.
Introduction
Within the last few decades, there has been significant progress in the treatment of specific types of human cancer, such as lymphomas, whereas other human cancers, including melanomas, pose a formidable challenge and have a poor prognosis despite aggressive treatment. Most of the agents used to treat cancer are DNA-damaging agents. The rationale for their use has been that during DNA replication, cells may be more sensitive to DNA damage. Recently, however, the increased sensitivity of cancer cells to DNA damage has been shown to involve the p53 tumor suppressor protein (1 , 2) . p53 is a sequence-specific DNA-binding transcription factor (3) . In response to DNA damage, p53 induces cell cycle arrest or apoptosis (4, 5, 6, 7) and thereby influences the outcome of therapy (1 , 2) . Many human cancers have developed genetic mutations that allow them to resist p53-dependent apoptosis. The most common mutations target the p53 gene itself, leading to expression of inactive p53 protein, which cannot induce apoptosis in response to DNA damage (3) .
Melanoma cells typically express wild-type p53 protein (8, 9, 10, 11, 12, 13, 14, 15) and would be expected to be sensitive to the DNA-damaging agents used for cancer therapy; however, these cells are extremely radioresistant (16 , 17) . There are several ways by which the function of wild-type p53 could be inhibited in melanoma. The defect could be downstream of p53. For example, high levels of Mdm2 protein could inhibit the ability of p53 to activate transcription, as occurs in sarcomas (18) . Alternatively, the defect could be upstream, in the signaling pathways that activate p53 in response to DNA damage. Consistent with this hypothesis, mutations in chk2/hcds1, a gene that encodes a DNA damage signaling kinase upstream of p53 (19, 20, 21, 22) , have been identified in a subset of families with the hereditary Li-Fraumeni cancer predisposition syndrome (23) .
The hypothesis that defective activation of wild-type p53 in response to DNA damage accounts for melanoma radioresistance can now be pursued because of the recent progress in our understanding of the molecular mechanisms by which DNA damage activates p53. Specifically, DNA damage leads to an increase in p53 protein levels (4 , 24) and to an increase in the functional activity of the p53 protein (25 , 26) . The molecular mechanism for increased p53 protein levels in response to exposure to IR3 involves a signaling pathway that includes the ATM and Chk kinases; ATM activates Chk1 and Chk2/hCds1, which in turn phosphorylate p53 on Ser-20 (19, 20, 21, 22 , 27) . The latter phosphorylation leads to dissociation of p53 from Mdm2 (21 , 28, 29, 30) , an intracellular protein whose normal function is to target p53 for degradation (31 , 32) . The mechanism for the increase in p53 functional activity in response to IR involves dephosphorylation of Ser-376 within the COOH terminus of p53. In the absence of DNA damage, Ser-376 and Ser-378 of p53 are phosphorylated; after exposure to IR, Ser-376 becomes dephosphorylated, creating a binding site for 14-3-3 proteins (33) . Interaction of p53 with 14-3-3 proteins allows p53 to transactivate target genes, whose protein products then induce cell cycle arrest or apoptosis.4 Unlike p53 stabilization, which is dependent on both the ATM and Chk kinases, Ser-376 dephosphorylation is ATM dependent, but Chk2/hCds1 independent (21) .
In an effort to better understand the molecular basis of melanoma radioresistance, we examined the regulation of p53 by DNA damage in human melanoma cell lines. Our analysis revealed an aberrant response, which may explain why melanoma cells are radioresistant.
Results
Constitutive Dephosphorylation of Ser-376 of p53 and Lack of
Interaction of p53 with 14-3-3 Proteins in Melanoma Cells.
The normal response of p53 to IR includes an increase in p53 protein
levels, dephosphorylation of Ser-376, and association of p53 with
14-3-3 proteins. As far as we could determine, normal melanocytes
exhibited a typical p53 response. IB of nuclear extracts with antibody
DO1 indicated that p53 protein levels increased about 3-fold 1 h
after irradiation. Furthermore, Ser-376 became dephosphorylated, as
indicated by increased immunoreactivity with PAb421, an antibody
specific for p53 that is not phosphorylated on Ser-376 (Fig. 1A)
. The association of p53 with 14-3-3 proteins after
exposure to IR could not be studied because of the overall low levels
of endogenous p53 in normal melanocytes.
|
. After irradiation, p53 levels were unchanged in one cell
line (WM1158) and increased about 3-fold in the other two cell lines
(1205LU and WM115). These results suggest that the DNA damage pathways
leading to increased p53 protein levels are mostly intact.
Dephosphorylation of Ser-376 was monitored by IB with antibody PAb421.
In all three cell lines, there was no change in the phosphorylation
state of Ser-376 in response to IR (Fig. 1C)
. Comparison of
the PAb421 immunoreactivities of wild-type p53 from melanocytes,
melanoma cell lines, and non-melanoma cell lines (U2OS osteosarcoma and
MCF7 breast adenocarcinoma cells) indicates that in melanoma cells, in
the absence of DNA damage, p53 is not phosphorylated on Ser-376 (Fig. 1
, B—D). Thus, whereas the normal response of
p53 to IR is dephosphorylation of Ser-376, in melanoma cells, Ser-376
is constitutively dephosphorylated.
Ser-376 is part of a recognition site within the COOH terminus of p53
for 14-3-3 proteins. The phosphorylation states of Ser-376 and Ser-378
within this site determine whether 14-3-3 proteins will bind to p53
(33)
. Because Ser-376 is aberrantly dephosphorylated in
melanoma cells, we examined whether p53 and 14-3-3 associate in these
cells. By coimmunoprecipitation analysis, we could detect no
interaction between the two proteins in both irradiated and
nonirradiated cells, whereas, as a positive control, p53 and 14-3-3
associated in irradiated U2OS osteosarcoma cells (Fig. 1E)
.
The absence of an interaction between p53 and 14-3-3 in melanoma cell
lines could not be attributed to obvious differences in p53 or 14-3-3
protein levels as compared with non-melanoma cell lines (Fig. 1E)
and implies that in melanoma cells, Ser-378, like
Ser-376, is dephosphorylated because phosphorylated Ser-378, in
conjunction with dephosphorylated Ser-376, would have created a high
affinity site for 14-3-3 proteins (33)
.
Intracellular Localization, Sequence-specific DNA Binding, and
Transcriptional Activities of Wild-Type p53 in Melanoma Cells.
Using mutant p53 proteins that fail to interact with 14-3-3, we have
recently established that the interaction between p53 and 14-3-3 does
not affect the intracellular localization of p53 or its ability to bind
sequence-specific oligonucleotides but is critical for its ability to
activate transcription of endogenous genes and induce cell cycle
arrest.4
In melanoma cells, wild-type p53 does
not bind to 14-3-3 proteins; therefore, we examined whether its
functional properties would be similar to those of p53 proteins with
mutant 14-3-3 recognition sites.
The intracellular localization of p53 was examined by
immunofluorescence using antibody DO1. In two melanoma cell lines that
express wild-type p53, WM1158 and 1205LU, the p53 protein was present
in the nucleus, and its localization was not affected by exposure of
the cells to IR (Fig. 2)
. Whereas some cells also showed cytoplasmic p53 localization (data not
shown), there were no cells that showed exclusively cytoplasmic
staining. Thus, the aberrant phosphorylation of p53 in melanoma
and the lack of interaction with 14-3-3 proteins did not lead to
exclusion of p53 from the nucleus.
|
. In response to irradiation, p53 DNA binding increased, paralleling
the increase in p53 protein levels (compare Figs. 1C
and 3
).
As expected, no DNA binding was observed using nuclear extracts of
melanoma cells that express mutant p53 (data not shown). From these
results, we conclude that wild-type p53 from melanoma cells can bind
avidly to sequence-specific oligonucleotides.
|
. Similar results were obtained with WM115 and WM1158
cells. In contrast, in melanocytes, expression of the p53 target genes
was significantly induced after Ad-p53 infection (Fig. 4A)
. Whereas it is formally possible that the inability of
wild-type p53 to induce expression of several of its target genes in
melanoma cells is due to independent mutations in the promoters of
these genes, our favored interpretation of the results is that the
transcriptional activity of wild-type p53 is compromised and that this
defect developed during melanoma progression, possibly as a way to
inactivate p53 function. Consistent with this interpretation, in
melanoma cells, in which p53 function was inactivated by mutations in
the p53 gene itself, Ad-p53 infection led to the
induction of p53 target genes (Fig. 4B)
. Furthermore, the
different responses among the cell lines could not be attributed to
differences in p53 protein expression, which was monitored by IB (data
not shown).
|
; data not shown): most of the cells had a 2 N DNA content
(G1); a smaller but significant fraction of cells
had a 4 N DNA content (G2-M phase);
and few cells had a DNA content greater than 2 N but less
than 4 N (S phase). Forty-eight h after exposure to IR, the
cell cycle profile suggested that the endogenous wild-type p53 protein
was functionally inactive. First, the fraction of cells with a 2
N DNA content decreased, whereas the fraction of cells with
a 4 N DNA content increased, indicating cell cycle arrest
in G2-M phase, but not in
G1. Second, a significant number of cells had an
8 N DNA content. These are cells that exited mitosis and
initiated a new round of DNA replication, although they failed to
undergo cytokinesis. This indicates a defect in p53 function because
cells with functional p53 do not undergo a new round of DNA replication
in the absence of cytokinesis (35, 36, 37, 38)
. Third, there were
no cells with a sub-G1 DNA content, which would
be expected if the endogenous wild-type p53 had induced apoptosis.
Fourth, the cell cycle profile was qualitatively identical to that of
melanoma cells with endogenous mutant p53 (Fig. 5)
.
|
; wild-type p53 overexpression did not inhibit the growth
of cultures of melanoma cell lines that express wild-type p53 (WM115
and 1205LU) but did inhibit the growth of cultures that express mutant
p53 (WM164 and WM852). To determine whether apoptosis underlaid the
inhibition of cell culture growth, the infected cells were examined for
signs of apoptosis using sub-G1 DNA content and
TUNEL assays. As observed with inhibition of culture growth, apoptosis
following infection with the adenovirus that expresses wild-type
p53 also correlated with p53 genotype (Fig. 6B)
. The resistance to apoptosis in melanoma cells with
endogenous wild-type p53 could not be attributed to lack of p53
expression by the adenoviral vector because Western blot analysis
revealed that infection of the melanoma cells with Ad-p53
resulted in p53 protein levels that were significantly elevated
compared with those of endogenous p53 (data not shown). Thus,
both endogenous and ectopically expressed wild-type p53 are
functionally defective in melanoma cells.
|
Most melanoma cells express high levels of the wild-type p53 tumor suppressor protein (39, 40, 41, 42) , which is a paradox because high levels of wild-type p53 are incompatible with tumor cell proliferation. We propose that this paradox can be resolved because the function of wild-type p53 protein is apparently compromised in melanoma cells. Specifically, in these cells, the endogenous wild-type p53 protein failed to induce cell cycle arrest or apoptosis in response to DNA damage, and ectopic wild-type p53 expressed by infection of melanoma cells with an adenoviral vector also failed to transactivate p53 target genes and promote apoptosis. These findings are consistent with a previous report showing decreased p53 transcriptional activity in melanoma cells (43) . The p53 functional defect was specific to melanoma cells that have endogenous wild-type p53. Ectopic wild-type p53 activated transcription and induced apoptosis when expressed in melanoma cells that have endogenous mutant p53. Furthermore, ectopic wild-type p53 was functionally active in melanocytes, from which melanoma cells develop. Thus, inactivation of p53 occurred during melanoma progression and was due to either mutation of the p53 gene or, more frequently, inhibition of the function of the wild-type p53 protein.
Wild-type p53 function involves two steps. First, upstream signaling pathways stabilize and activate the p53 protein in response to DNA damage or other forms of cellular stress. Activated p53 then binds to regulatory sequences of its target genes and activates their expression. Either step may be defective in melanoma cells. Several proteins have been described that regulate p53 at the level of the second step. Mdm2 inhibits p53 transactivation when overexpressed and is clinically relevant in sarcomas (18 , 44) . However, melanoma cells, with few exceptions, do not express high levels of Mdm2 (45) . Two other proteins, IFN regulatory factor 1 and p33ING1, physically associate with p53 and are required for p53-mediated transactivation (46 , 47) . However, mutations of the genes encoding these two proteins are not frequent in human cancer (48) , and their role in melanoma development is unclear. Another protein that could account for the low transcriptional activity of p53 in human melanoma cells is p14ARF, one of the two protein products of the INK4a locus (49) . p14ARF inhibits the Mdm2-dependent degradation of p53 as well as the ability of Mdm2 to suppress the transcriptional activity of p53 (50 , 51) . Thus, in melanoma cells, where INK4a mutations are exceedingly common (52, 53, 54, 55) , loss of p14ARF expression could very well explain the decreased p53 transcriptional activity. The only caveat of this model is that it predicts low level expression of p53 protein, whereas the inverse is typically true in melanoma cells (39, 40, 41, 42) .
Defective activation of wild-type p53 after DNA damage is our preferred mechanism for suppression of p53 function in melanoma cells. The response of p53 to DNA damage involves an increase in p53 protein levels due to stabilization of the protein and an increase in p53 functional activity (4 , 24, 25, 26) . Most likely there is no defect in regulation of p53 stabilization in response to DNA damage because p53 was stabilized in response to IR in two of the three melanoma cell lines examined, and the steady-state levels of p53 were very high. In contrast, regulation of the functional activity of p53 appears to be defective. In normal cells, regulation of p53 function by IR involves dephosphorylation of Ser-376 and association of p53 with 14-3-3 proteins (33) . In melanoma cells, wild-type p53 had Ser-376 constitutively dephosphorylated and failed to interact with 14-3-3 proteins in response to DNA damage. The lack of interaction between p53 and 14-3-3 can lead to a p53 functional defect, as suggested by analysis of mutant p53 proteins that do not bind 14-3-3 in osteosarcoma cells.4 Specifically, these mutant p53 proteins retain the ability to bind sequence-specific DNA in vitro and localize to the nucleus when expressed in cells, but they fail to activate transcription of endogenous p53 target genes and induce cell cycle arrest and apoptosis. These properties are very similar to the properties of wild-type p53 in melanoma cells.
The mechanism by which 14-3-3 modulates the activity of p53 is as yet unclear. 14-3-3 can affect the sequence-specific DNA binding activity of p53 (33) . Thus, although wild-type p53 from melanoma cells bound DNA efficiently in vitro, its ability to bind to the promoters of its target genes in vivo may be compromised in the absence of 14-3-3. Alternatively, 14-3-3 proteins may modulate the transcriptional activity of p53. In plant cells, 14-3-3 proteins augment the transcriptional activity of several sequence-specific transcription factors. In some cases, the mechanism involves translocation of the transcription factor from the cytoplasm to the nucleus; in other cases, the mechanism involves recruitment of basal transcription factors (56, 57, 58) . The latter mechanism may be more relevant for p53 because 14-3-3 proteins do not affect the intracellular localization of p53 in osteosarcoma cells,4 and p53 was nuclear in the melanoma cell lines examined by us.
Whereas the most common mechanism leading to p53 inactivation in human cancer is mutations targeting the p53 gene, it is becoming increasingly clear that in some tumors, the defect lies in the signaling pathways that regulate p53 in response to DNA damage. For example, the genes encoding the kinases ATM and Chk2/hCds1 are mutated in human cancer. ATM is required for stabilization and functional activation of p53 in response to IR and is mutated in patients with ataxia-telangiectasia, who have an increased incidence of cancer (59) . Chk2/hcds1, which is required for p53 stabilization in response to IR, UV light, and replication blocks (20, 21, 22) , is mutated in some families with the Li-Fraumeni cancer predisposition syndrome (23) . According to this precedence, we propose that in melanoma cells, the regulation of p53 in response to DNA damage is defective. The defect leads to lack of interaction between p53 and 14-3-3 proteins, but its precise nature remains to be resolved. Because the 14-3-3 binding site on p53 includes Ser-376 and Ser-378 and binding occurs when Ser-376 is dephosphorylated and Ser-378 is phosphorylated (33) , the defect may involve the kinase(s) and phosphatase(s) that target Ser-376 and Ser-378. Irrespective of its precise nature, a defect in p53 function could contribute to the high radioresistance of melanoma in the clinic (16 , 17) .
Materials and Methods
Cell Lines, Viral Vectors, and Antibodies.
The melanoma cell lines used in this study were established from
primary and metastatic lesions (60)
and maintained in
MCDB153/L15 medium containing insulin and 2% FCS. The p53
genotype of these cell lines, which was known from the literature, was
confirmed by sequencing cDNA amplified by PCR using
p53-specific primers. Isolation and maintenance of normal
melanocytes from the neonatal foreskin have been described previously
(60)
. An adenoviral vector containing the p53
gene under the control of the cytomegalovirus promoter
(Ad-p53) has been described previously (61)
.
This adenoviral vector and a vector containing the lacZ
reporter gene (Ad-lacZ) were produced in human 293 cells,
purified by double CsCl2 centrifugation, and
titrated according to standard procedures (62)
. Antibodies
to p53 (PAb421 and DO1), p21 (EA10), and 14-3-3 (K19) were obtained
from Calbiochem (San Diego, CA) and Santa Cruz Biotechnology (Santa
Cruz, CA).
p53 Protein Levels, Ser-376 Phosphorylation State, and DNA
Binding Activity.
Nuclear extracts were prepared as described previously
(33)
. p53 protein levels were determined by IB of the
extracts with antibody DO1. The phosphorylation state of Ser-376 was
determined by IB with antibody PAb421, which recognizes p53 only when
Ser-376 is dephosphorylated. For DNA binding, the nuclear extracts were
incubated with 32P-labeled oligonucleotide Ep21,
which contains the p53 response element in the p21/cip1/waf1
gene, and analyzed for p53 DNA binding by native PAGE
(63)
.
Immunofluorescence.
Melanoma cells were grown on glass coverslips, fixed in 1%
paraformaldehyde, permeabilized with Triton X-100, and incubated with
antibody DO1. After washing, the cells were incubated with a Texas
red-conjugated secondary antibody and stained with
4',6-diamidino-2-phenylindole.
p53 Transactivation.
Melanoma cells infected with Ad-p53 or Ad-lacZ at
a MOI of 10 were harvested 24 h after infection, and total RNA was
isolated using the Trizol reagent (Life Technologies, Inc.,
Gaithersburg, MD). The RNA (15 µg/lane) was separated by
electrophoresis on 1% agarose gels, transferred to nitron membranes,
and probed for expression of p53 target genes.
Cell Growth and Apoptosis.
Cell growth was assayed by [3
H]thymidine
incorporation. Briefly, the melanoma cells were seeded in 96-well
plates at 3 x 104
cells/well. Twenty-four h
later, the cells were treated in quadruplicate with Ad-lacZ
or Ad-p53 at various levels of MOI for 3 h. Sixty h
later, the cells were pulsed for 4 h with
[3
H]thymidine, harvested on GF/C Whatman filter
paper, and counted for radioactivity. The experiments were repeated
twice. Apoptosis was examined in response to irradiation or in response
to infection with Ad-p53 or Ad-lacZ at a MOI of
10 using the TUNEL assay or propidium iodide staining (64
, 65)
. For the TUNEL assay, the cells were harvested in 1%
paraformaldehyde in PBS to immobilize the apoptotic DNA and then fixed
with 80% ethanol to permeabilize the cells. The apoptotic DNA
fragments were end-labeled with fluorescein-labeled dUTP using terminal
deoxynucleotidyl transferase and analyzed by flow cytometry. For the
propidium iodide assay, the cells were fixed in 80% ethanol, stained
with propidium iodide in the presence of RNase A, and analyzed by flow
cytometry.
Acknowledgments
We thank David Elder, Donna George, Dorothee Herlyn, Daniel Scolnick, and Elena Stavridi for helpful discussions and reagents.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported by USPHS Grants CA25874 (to M. H. and
T. D. H.) and CA09171 (Wistar Institute Training Grant) awarded by
the National Cancer Institute, Department of Health and Human
Services. 
2 To whom requests for reprints should be
addressed, at the The Wistar Institute, 3601 Spruce Street,
Philadelphia, PA 19104-4268. E-mail: halazonetis{at}wistar.upenn.edu
3 The abbreviations used are: IR, ionizing
radiation; TUNEL, terminal deoxynucleotidyl transferase-mediated nick
end labeling; ARF, alternate reading frame; IB, immunoblotting; MOI,
multiplicity of infection.
4 N. H. Chehab and T. D. Halazonetis,
manuscript in preparation.
Received for publication 5/ 1/00. Revision received 7/13/00. Accepted for publication 7/17/00.
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) or p53 (
) at the indicated
MOIs. After 60 h, the cells were incubated with tritiated
thymidine, and the amount of incorporated label was determined 4 h
later. B, apoptosis. Melanoma cells 1205LU (endogenous
wild-type p53) and WM852 (mutant p53) were infected with adenoviruses
expressing lacZ (Ad-lacZ) or wild-type
p53 (Ad-p53) at a MOI of 10, and the
percentage of apoptotic cells was determined 24 h later by
monitoring the fraction of cells with a sub-G1 DNA content
after propidium iodide staining or by the TUNEL assay.