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NEOPLASIA
From the Molecular Pathology Program, Centro Nacional
de Investigaciones Oncológicas Carlos III (CNIO); Department of
Immunology and Oncology, CNB/CSIC-Pharmacia; Cancer Epidemiology
Service, Centro Nacional de Epidemiología, Instituto de Salud
Carlos III, Madrid, Spain.
p14ARF, the alternative product from the human
INK4a/ARF locus, antagonizes Hdm2 and mediates p53
activation in response to oncogenic stimuli. An immunohistochemical
study of p14ARF expression in 74 samples of aggressive
B-cell lymphomas was performed, demonstrating an array of different
abnormalities. A distinct nucleolar expression pattern was detected in
nontumoral tissue and a subset of lymphomas (50/74). In contrast, a
group of cases (8/74) showed absence of p14ARF expression,
dependent either on promoter hypermethylation or gene loss.
Additionally, 16 out of 74 cases displayed an abnormal nuclear
p14ARF overexpression not confined to the nucleoli, as
confirmed by confocal microscopy, and that was associated with high
levels of p53 and Hdm2. A genetic study of these cases failed to show any alteration in the p14ARF gene, but revealed the
presence of p53 mutations in over 50% of these cases. An increased
growth fraction and a more aggressive clinical course, with a shortened
survival time, also characterized the group of tumors with
p14ARF nuclear overexpression. Moreover, this
p14ARF expression pattern was more frequent in tumors
displaying accumulated alterations in the p53, p16INK4a,
and p27KIP1 tumor supressors. These observations, together
with the consideration of the central role of p14ARF in
cell cycle control, suggest that p14ARF abnormal nuclear
overexpression is a sensor of malfunction of the major cell cycle
regulatory pathways, and consequently a marker of a high tumor aggressivity.
(Blood. 2002;99:1411-1418) The 2 major tumor suppressor pathways, represented
by the proteins p16INK4a-CDK4/6-Rb and
p14ARF-Hdm2-p53,1 are inactivated in most
human cancers. p53 is a transcription factor that induces cell cycle
arrest and/or apoptosis in response to a variety of stimuli (eg, DNA
damage, hyperproliferative signals).2 p53 is negatively
regulated by Hdm2 through a multiple mechanism: Hdm2 binds to the
transactivation domain of the p53 tetramer, inhibiting p53
transcriptional activity3,4; in addition, Hdm2 functions as
an E3-ubiquitin ligase which targets p53 for nuclear export and
proteasomal degradation.5-8 Hdm2 is itself a
p53-responsive gene, thus establishing a feedback loop through which
p53 regulates its own activity and turnover.9
The atypical structure of the INK4a/ARF locus in 9p21
encodes 2 unrelated tumor suppressor proteins, p16INK4a and
p14ARF (the human counterpart of murine
p19ARF).10,11 These are specified by different
first exons that are spliced to a common second exon translated in
alternative reading frames, their expression being controlled by
independent promoters. p16INK4a, a specific inhibitor of
cyclin D-dependent kinases, contributes to G1 arrest by blocking Rb
phosphorylation.1 On the other hand, p14ARF
interferes with all of the known functions of Hdm2 (eg, direct inhibition of p53-mediated transactivation,12,13 ubiquitin ligase activity,14 and nuclear export of
p5315,16), possibly through induction of Hdm2
degradation,17 indirectly leading to an increase in the
activity and stability of p53. p14ARF is induced by
inappropriate hyperproliferative signals (such as
myc,18 E2F-1,19
ras,20 E1A,21
v-abl22) and mediates p53 activation in
response to oncogenic stimuli. Specifically, responsiveness of the
p14ARF promoter to E2F-1 makes p14ARF a nexus
between the Rb and p53 pathways. p53 suppresses p14ARF
expression through a poorly understood mechanism, which generates an
additional regulatory circuitry.13,23
p14ARF is a highly basic protein that localizes to the
nucleolus.10,12,24 When induced, p14ARF binds
to Hdm2, thereby allowing p53 to stabilize and accumulate in the
nucleoplasm. Classically, this p14ARF-Hdm2 binding has been
assumed to take place in the nucleolus,16 although
antagonization of Hdm2 by p14ARF independently of nucleolar
localization has recently been reported.25 In addition, it
has been suggested that p14ARF-p53 direct binding without
requirement for Hdm2 as a bridging molecule is also
possible,23 although the functional implications of this
interaction remain unknown.
Both human and murine p14ARF contact the central acidic
domain of Hdm2 through 2 independent binding sites.26-28
Additionally, nucleolar localization sequences (NrLS) have been mapped
in exons 1 In human tumors, the p53 gene is inactivated by mutation in more than
50% of cases; in a high proportion of the rest, the p53 pathway would
be expected to be disrupted by Hdm2 amplification or
p14ARF loss. In some cancers, the frequency of
p14ARF alteration is remarkably high; deletions affecting
the 9p21 region (eg, in the cases of glioblastoma and
astrocytoma30,31) and hypermethylation of CpG islands in
the p14ARF promoter (eg, in the case of gastric
cancer32) are the main inactivation mechanisms. Point
mutations are infrequent and usually also affect p16INK4a.
In other neoplasias, p14ARF loss appears to be a rarer
event. Nevertheless, most of the information available is derived from
analysis at the gene level, whereas little is known about the
expression level and distribution of the protein in different tumors.
In aggressive B-cell non-Hodgkin lymphomas (NHLs), the frequency of p53
mutation is modest (~20%),33,34 whereas amplification of the 12q14 region (where the Hdm2 gene is located) has not
been detected.35 Therefore, our purpose was to investigate
the expression pattern of the p14ARF protein and its
subcellular localization, the possible presence of genetic or
epigenetic alterations in p14ARF, and the relationship
between the status of p14ARF and the major tumor suppressor
pathways in a group of large B-cell lymphoma and Burkitt lymphoma.
Tissue samples
Cell lines
For immunostaining, cells were harvested by centrifugation, washed with cold phosphate buffered saline (PBS), cytospun onto poly-L-lysine-coated slides, and fixed in ethanol/acetone 1:1. Mutational analysis Exons 5 to 8 of the p53 gene and exons 1 and 2 of the
p14ARF gene were amplified from genomic DNA extracted from
tissue samples and cell lines, using previously described primers and
conditions.31,34,37 Polymerase chain reaction (PCR)
products were purified using the Microcon PCR system (Millipore,
Bedford, MA). Direct sequencing of purified PCR products was performed
with an automated DNA Sequencer ABI PRISM 3700 Genetic Analyzer
(Applied Biosystems, Weiterstadt, Germany). Results for p53 and
p14ARF exon 2 mutations in tumor samples have been
previously published.33,38
Allelic loss Homozygous deletions affecting p14ARF in cell lines were confirmed by simultaneous amplification of p14ARF exon 2 and p53 exon 8 (multiplex PCR). Results for homozygous or hemizygous deletions in the 9p21 region in the series of NHLs have been published as part of previous studies concerning p16INK4a status.33,38Analysis of p14ARF promoter hypermethylation Methylation-specific PCR (MSP)39 assays were performed to determine the methylation status of the CpG islands of the p14ARF promoter in tissue samples and cell lines. Briefly, 1 µg of denatured genomic DNA was modified by reaction with sodium bisulfite under conditions that convert unmethylated cytosines to uracils. Modified DNA was purified using the Wizard DNA Clean-Up system (Promega, Madison, WI). Modification was completed by NaOH 0.3 M treatment for 5 minutes at room temperature, followed by ethanol precipitation.A quantity of 50 ng bisulfite modified DNA was amplified using p14ARF unmethylated-specific and methylated-specific primers.40 The Hodgkin disease-derived L-540 cell line was used as a positive control of p14ARF methylation41; DNA from nontumoral samples was included as a negative control. Methylation of the p14ARF promoter was detected by the amplification of a 122-bp fragment with the methylated-specific primers. Antibodies The following primary antibodies were used for immunohistochemistry: goat polyclonal anti-p14ARF C-18 (Santa Cruz Biotechnology, Santa Cruz, CA); mouse monoclonal anti-Hdm2 Ab-1 2IF-2 (Oncogene, Darmstadt, Germany); mouse monoclonal anti-p53 DO-7 (Novocastra, Newcastle upon Tyne, United Kingdom). Proliferation index was evaluated by the expression of the nuclear antigen Ki67, detected with the MIB1 monoclonal antibody (Novocastra).Immunofluorescence was performed with the same antibodies, with the exception of Hdm2, which was detected with the SMP14 monoclonal antibody (Santa Cruz Biotechnology). Mouse monoclonal anti-C23 MS-3 (Santa Cruz Biotechnology) was used as a nucleolar marker.42,43 Immunohistochemistry Immunohistochemical techniques were performed on paraffin-embedded tissue sections or cytospin preparations of the cell lines using an initial heat-induced antigen retrieval step (slides were heated in a pressure cooker for 3 minutes in a 0.01 M solution of sodium citrate prior to incubation with the antibodies).After incubation with the primary antibodies, immunodetection was performed with biotinylated antimouse or antigoat secondary antibodies as appropriate, followed by peroxidase-conjugated streptavidine (DAKO, Carpinteria, CA) with diaminobenzidine chromogen as substrate. Immunostaining was performed with the Techmate 500 (DAKO) automatic immunostaining device. Incubation omitting the specific antibody was used as a control of the technique. p53 and Hdm2 expression were scored semiquantitatively and expressed as the nearest tenth percentile. Ki67 expression was quantified by scoring up to 200 tumoral cells in representative areas. Immunofluorescence and confocal microscopy Double fluorescent immunolabeling was performed on 3-µm-thick tissue sections mounted on poly-L-lysine-coated slides. After antigen retrieval and simultaneous incubation with the 2 primary antibodies, slides were washed and incubated with the secondary antibodies Cy3-conjugated donkey anti-goat (Jackson Immunoresearch, Baltimore, MD) and Alexa 488-conjugated rabbit anti-mouse (Molecular Probes, Eugene, OR). Nuclei were stained with TO-PRO-3 (Molecular Probes). The slides were mounted with VectaShield (Vector, Burlingame, CA) and examined with a TCS NT laser scanning confocal microscopy system (Leica Microsystems, Wetzlar, Germany). The series of images was processed with the software package provided (Leica Microsystems) and Adobe Photoshop 5.5.Statistical analysis The relationship between p14ARF and other variables (p53, Hdm2, Ki67) was examined using the Kruskal-Wallis test. In order to assess the specific association between p14ARF and Ki67, a multivariate analysis was performed including those markers significantly related to Ki67 levels as revealed by the univariate analysis. Since neither Ki67 nor its log-transformed values were normally distributed, as assumed by standard multiple regression techniques, a median regression analysis44 was performed. This method compares the median instead of the mean of each category.The prognostic value of p14ARF and all other markers was evaluated by standard survival analysis, using Kaplan-Meier and Cox regression. Hazard ratios were computed for each marker, adjusting for International Prognostic Index (IPI) and histology. Statistical analyses were performed using the STATA and SPSS software packages.
Analysis of p14ARF, Hdm2, and p53 expression Cell lines.
p14ARF expression was analyzed by immunostaining in several
human lymphoid cell lines in which p53 and p14ARF gene
status were also determined (Figure 1;
Table 1).
Normal human tissues.
In nontumoral lymphoid tissue (tonsils and reactive
lymphadenitis), reactive cells showed distinct nucleolar
p14ARF immunostaining (Figure
2A). Staining for Hdm2 (Figure 2B) and p53 (Figure 2C) revealed weak nucleoplasmic expression of both proteins
in proliferating cells within the germinal centers. No Hdm2 nucleolar
staining was observed in normal cells.
Aggressive B-cell lymphomas. p14ARF expression was analyzed in a series of 74 cases of NHLs including 55 DLBCL, 7 FL-3, and 12 BL. In these samples, an internal control of the technique was provided by the nucleolar staining of small lymphocytes, macrophages, and some endothelial cells. Concerning the p14ARF expression pattern in tumoral cells, the following situations were observed: (1) p14ARF loss in a subset of NHLs. A small number of cases (8/74; Table 2) showed no appreciable p14ARF staining in large cells (Figure 2D), whereas nucleolar staining was observed in benign lymphocytes. The predominant phenotype in this subset of cases was characterized by a low level of expression of both Hdm2 (Figure 2E) and p53 (Figure 2F). Of these cases, 3 could be sequenced for p14ARF, but no mutations were found. In 1 of 3 cases (case 19), silencing of the p14ARF gene was associated with promoter hypermethylation coupled with an LOH (loss of heterozygosity), which could affect the unmethylated allele. A homozygous deletion within the 9p21 region was detected in another case (case 47). The p53 gene was wild-type in the 4 cases sequenced. (2) Nucleolar p14ARF expression. Nucleolar p14ARF staining in tumoral cells was the most frequently observed expression pattern (50/74 cases). The intensity of staining was variable, ranging from weak to strong nucleolar staining in most large cells (Figure 2G). The staining for Hdm2 (Figure 2H) and p53 (Figure 2I) was always nucleoplasmic and of variable intensity, often with distinct nucleolar exclusion. Nucleolar accumulation of Hdm2 was never observed. No alterations in the p14ARF sequence (a nucleotide change in exon 2, detected in one case,33 does not affect the p14ARF protein) or promoter hypermethylation were detected in any of the 42 cases analyzed in this group; 4 cases showed LOH in 9p21. p53 mutations were detected in a minority of cases (1 nonsense and 3 missense mutations out of 42 samples). (3) p14ARF nuclear overexpression. A subset of NHLs (16/74) showed an atypical p14ARF expression pattern characterized by intense nuclear staining that was not confined to the nucleolus (Figure 2J). Since mutations affecting the NrLS of p14ARF have been described as partially or totally impeding the correct localization of this protein,29 both exons of the p14ARF gene were sequenced in 9 of 16 cases. However, no alterations were detected. Allelic loss and promoter hypermethylation analyses, performed in the same samples, also yielded negative results. This group of cases (Table 3) was characterized by high p53 and Hdm2 levels of expression (Figure 2K-L). When compared with the rest of the series, a statistically significant relationship was found between p14ARF nuclear overexpression and higher expression of both p53 and Hdm2 (Kruskal-Wallis test, P = .001 for p53 and P = .010 for Hdm2). p53 was either wild-type or mutated in this subset of cases, but the frequency of p53 mutation was significantly higher (5/9 sequenced cases) when compared with the rest of the series (Fisher exact test, P = .005). There were no significant differences in the distribution of the cases with p14ARF nuclear overexpression between the categories defined by histologic diagnosis (BL vs non-BL; Fisher exact test, P = .715) or IPI (Pearson chi square: 0.360, P = .948).
Analysis of subcellular localization In order to characterize more precisely the subcellular localization of the p14ARF, Hdm2, and p53 proteins, 5 tumor samples with different patterns of expression of p14ARF were analyzed by confocal microscopy. Double fluorescent immunolabeling was performed for p14ARF and either C23 (nucleolar marker), p53, or Hdm2.Existence of a nucleoplasmic fraction of p14ARF was
effectively observed in cases with abnormal p14ARF nuclear
overexpression, as demonstrated by double labeling with C23 (Figure
3A). Although nucleolar exclusion was not
observed, part of the signal was clearly extranucleolar. Nucleoplasmic
p14ARF was restricted to the large tumoral cells, whereas
small lymphocytes showed distinct nucleolar p14ARF
staining. This pattern contrasts with the more typically observed situation in both normal and tumoral cells, in which p14ARF
was confined to the nucleolus (Figure 3B). C23 characteristic nucleolar
staining demonstrates that the integrity of the nucleolus was always
preserved, and anomalous localization of p14ARF was not a
consequence of structural alterations of the nucleolus.
Double labeling for p14ARF-p53 revealed a partial colocalization of these proteins in the nucleoplasm, suggesting the existence of a fraction of nucleoplasmic p14ARF not bound to p53 (Figure 3C). This colocalization was absent in samples exhibiting p14ARF nucleolar localization (Figure 3D). A striking pattern was observed for Hdm2, in that it was not found to colocalize with nucleoplasmic or nucleolar p14ARF; in fact, these signals usually appeared in alternative cells, and Hdm2 never accumulated in the nucleolus (Figure 3E-F). Clinical and biologic significance of p14ARF nuclear overexpression Since p14ARF has been reported as accumulating in response to oncogenic stimuli,18-22 we examined whether p14ARF nuclear overexpression had any correspondence with the biology of the tumor or clinical outcome of the patients.To this end, Ki67 expression was quantified in 70 of 74 cases (range:
32%-100%, median: 82%) as a measure of proliferation index.
Univariate analysis showed a strong association between p14ARF nuclear overexpression and higher Ki67 levels
(Kruskal-Wallis test, P = .002). Histology (BL vs non-BL)
and p53 and Hdm2 expression were also significantly related to Ki67
(Table 4). However, after performing a
multivariate analysis that included the variables found to be
significant in the univariate analysis, the only variable that retained
statistical significance, in addition to histology, was
p14ARF nuclear overexpression (P = .035).
To test this possible association between p14ARF nuclear
overexpression and tumor aggressivity further, an overall survival
analysis was performed for the subset of cases (51/74) for which
clinical follow-up information during at least 60 months was available. A worse prognosis was observed for the cases with nuclear
p14ARF when compared with the rest of the series
(P = .060, crude analysis) (Table 5); this
relationship proved to be statistically significant after adjusting the
results by IPI and histology (P = .043; Figure 4A). Since the small group of cases in
which p14ARF expression was not detected may include
aggressive tumors in which p14ARF overexpression is impeded
by alterations at the gene level, the cases lacking p14ARF
expression were excluded from the analysis and overall survival of the
patients overexpressing p14ARF was compared with that of
the cases showing a nucleolar expression of the protein; again,
differences were found to be significant (P = .037).
p14ARF overexpression was also a negative predictor of survival when only DLBCLs were considered (P = .047, crude analysis); however, the reduced number of BL cases prevented us from performing informative statistical analyses for this histologic class as a separate group. When cases were grouped according to IPI and overall survival was analyzed separately for each group, a similar association between p14ARF nuclear overexpression and prognosis was found for the category of medium IPI (2-3) (P = .055); analyses were not considered informative for the groups of low (0-1) or high (4-5) IPI due to the reduced number of events in the first case and the small number of patients with p14ARF nuclear overexpression (2 patients) in the second. In a previous report33 we analyzed different alterations in tumor suppressor pathways in a group of NHLs that included 51 of the samples described here. For these cases, information was available concerning p53 mutations, p16INK4a inactivation by promoter hypermethylation, deletion, or mutation, and p27KIP1 overexpression, presumably reflecting its inactivation by CDK4-cyclin D3 binding.45 First, the relationship between p14ARF nuclear overexpression and each of these individual alterations was analyzed. As mentioned above, there was a strong association between p53 mutation and p14ARF nuclear overexpression (P = .005). Presence of nuclear p14ARF was also more frequent in tumors overexpressing p27KIP1, although this did not reach statistical significance (Fisher exact test, P = .118). An association between p16INK4a alterations and
p14ARF overexpression was not found when all the possible
mechanisms of inactivation of p16INK4a were considered as a
whole. However, this may be due to the fact that LOHs or homozygous
deletions at 9p21 are likely to affect both genes simultaneously,
preventing p14ARF from being overexpressed. Effectively,
when LOHs were excluded and only p16INK4a promoter
hypermethylation Finally, we analyzed the frequency of p14ARF abnormal overexpression as a function of the number of accumulated alterations in p53, p16INK4a, and p27KIP1. As shown in Figure 4B, the proportion of cases with anomalous p14ARF expression increases with the number of cell cycle defects, this relationship being statistically significant (Pearson chi square: 10.233, P = .017). Interestingly, p14ARF nuclear overexpression was not observed in any of the 19 cases where no alterations in the p53 and Rb pathways were found.
Information concerning regulation of the p14ARF-Hdm2-p53 pathway is mainly derived from in vitro studies and animal models, whereas a comprehensive analysis of its significance in human tumors has been hampered by technical considerations such as the lack of appropriate antibodies for detecting the p14ARF protein. We have chosen NHL as a model, since it features tumors with a high growth fraction in which p53 mutations are present in only a small proportion of cases, and therefore may be useful for revealing the complexity of molecular alterations taking place in this pathway. Unlike what has been found in other neoplasias, molecular alterations
resulting in loss of p14ARF expression are rare in NHLs.
Hypermethylation of p14ARF promoter and deletions within
the 9p21 region, the 2 main mechanisms for p14ARF
silencing, have been detected in our series of cases only
exceptionally. This situation sharply contrasts with that observed in
lymphoid cell lines, in which cancellation of the p53 pathway by
alternative In contrast with the exclusively nucleolar localization of p14ARF observed in normal cells and most in vitro models, abnormal p14ARF nucleoplasmic accumulation has been found in a significant number of aggressive NHLs. It does not seem likely that this is a consequence of nonspecific staining, as a distinct nucleolar signal has been obtained with the same antibody and under the same conditions in nontumoral tissue, many NHL samples, and several lymphoid cell lines. Moreover, no staining (either nucleolar or nucleoplasmic) has been detected in cell lines with p14ARF silencing by homozygous deletion (Figure 1). p14ARF-C23 double immunolabeling has demonstrated that our observations are not an artifact related to loss of nucleolar integrity. We have also ruled out the possibility that nucleolar distribution is impeded by mutations affecting the p14ARF NrLS, since the p14ARF gene has been found to be wild-type in all the cases that have been sequenced. The significance of this findings is enhanced by the observation that p14ARF atypical overexpression defines a group of lymphomas characterized by higher aggresivity. A strong association exists between p14ARF nuclear overexpression and higher proliferation index, which remains significant even in a multivariate analysis including relevant variables such as p53, Hdm2, and histologic diagnosis. A similar correlation existed at the prognostic level, since cases with abnormal p14ARF overexpression were also characterized by a shorter overall survival. An explanation for these findings can be found in the consideration of
the central role of p14ARF in cell cycle control, as a
nexus between the major tumor suppressor pathways. Thus, the
p14ARF promoter has an E2F-1 binding site which
"senses" oncogenic stimuli transduced through the Rb
pathway19; p14ARF is also induced by typical
oncogenes (myc, ras, viral genes), and negatively
regulated by p53 (Figure 5A). With the
exception of alterations affecting p14ARF itself, which we
have shown to be very rare in NHLs, virtually every cancer-related
defect in these pathways (Figure 5B) should result in
p14ARF upregulation: alterations of the Rb pathway such as
p16INK4a inactivation or cyclin D overexpression,
deregulation of oncogenes (eg, myc in BL), and p53
inactivation by mutation or by Hdm2 overexpression (resulting in
disruption of the p53-p14ARF negative feedback loop).
Therefore p14ARF should integrate all these stimuli, its
level of expression being a measure of the accumulation of alterations
in different points of the cell cycle, and consequently a marker of
tumor aggressivity. Consistent with this hypothesis, p14ARF
nuclear overexpression is a more frequent finding in tumors displaying simultaneous inactivation of several major tumor suppressors (p53, p16INK4a, p27KIP1), in addition to correlate
with some of these alterations, taken individually. The postulation of
an overexpressed nuclear p14ARF as a surrogate of a highly
deregulated cell cycle is consistent with the higher aggressivity
(as measured by proliferation index and worse prognosis) observed
in this group of lymphomas.
Even if p14ARF overexpression is a consequence of cell cycle malfunction, the atypical nuclear localization of the protein remains an intringuing finding. Results of confocal microscopy suggest that p14ARF nucleoplasmic accumulation is only partially dependent on p53 binding and probably independent of Hdm2 binding. A recent report has suggested that the predominant p14ARF nucleolar accumulation is accompanied by an usually undetectable nucleoplasmic fraction which could be responsible for p53 activation.25 Consistent with this model, it would be expected that under conditions of massive p14ARF induction the nucleoplasmic fraction would become detectable, as could be the case for a group of aggressive lymphomas. Our findings may help to resolve the controversy concerning Hdm2 and p14ARF subcellular localization, both in normal and tumoral cells. Thus, the presence of Hdm2 and p14ARF seems to be mutually exclusive, as shown by the lack of nucleolar Hdm2 staining in cells expressing nucleolar p14ARF, and the absence of Hdm2 in cells overexpressing nuclear p14ARF. This suggests that p14ARF-Hdm2 complexes, if they exist, should have a short half-life, dependent on a rapid Hdm2 degradation induced after p14ARF binding.
We are indebted to A. I. Sáez for her invaluable help with the statistical analysis, and to I. Fernández and M. J. Acuña for their expertise and excellent technical assistance with molecular and immunohistochemical assays, respectively. We also thank Dr Juan Carlos Martínez-Montero for his kind collaboration in the initial stages of the immunohistochemical analysis.
Submitted June 25, 2001; accepted October 11, 2001.
Supported by grants from the Fondo de Investigaciones Sanitarias (FIS 98/993), Ministerio de Sanidad y Consumo, Comisión Interministerial de Ciencia y Tecnología (1FD97-0431), Comunidad Autónoma de Madrid (08.1/0028/2000 1) and Ministerio de Ciencia y Tecnología (SAF2001-0060), Spain. A.S.-A. is supported by a grant from the Spanish National Cancer Center.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Margarita Sánchez-Beato, Molecular Pathology Program, Centro Nacional de Investigaciones Oncológicas Carlos III (CNIO), Ctra Majadahonda-Pozuelo, Km 2, 28220 Majadahonda, Madrid, Spain; e-mail: msbeato{at}cnio.es.
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Top
Abstract
Introduction
plus a single mutation that did not affect
p14ARF