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Cancer Research Unit, The Medical School, University of Newcastle, NE2 4HH [D. A. T., J. L.]; Departments of Child Health [D. A. T., A. D. J. P.] and Pathology [A. J. M.], Royal Victoria Infirmary, Newcastle upon Tyne, NE1 4LP; and Department of Biochemistry and Genetics, University of Newcastle, NE2 4AA [N. B.], United Kingdom
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ABSTRACT |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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An important mechanism of chemo- and radioresistance in many tumor cells is an abnormality in the p53 tumor suppressor gene pathway (5) . The p53 gene, located on the short arm of chromosome 17 at p13, is the most frequently mutated gene in human cancer. A mutation of one allele is often accompanied by loss of the second allele (loss of heterozygosity). p53 is a nuclear phosphoprotein induced in response to cellular stress such as DNA damage from radiation or alkylating agents and binds DNA in a sequence-specific manner to activate the transcription of a number of genes including p21WAF1, MDM2, and BAX (6) . WAF1 inhibits G1 cyclin-dependent kinases blocking cell cycle progression from G1 into S phase. MDM2 binds to p53 and blocks its ability to function as a transcription factor, so creating an autoregulatory feedback loop to tightly regulate p53 levels (5) . Tumors with mutant p53 cannot usually bind to DNA and up-regulate MDM2. Consequently, there is a lack of MDM2 to bind to p53 and target it for ubiquitin-mediated degradation, resulting in p53 accumulation.
p53 can also respond to cellular stress by inducing apoptosis. This may be transcriptionally dependent or independent. In some cell types p53 transcriptionally induces BAX, a proapoptotic gene that forms mitochondrial pores leading to cytosolic release of cytochrome c, which activate caspases and leads to apoptosis (6) . BAX may also form heterodimers with BCL2, an antiapoptotic membranous protein, which may be transcriptionally repressed by p53. Whether a cell undergoes growth arrest or apoptosis is dependent on the cell type, phase of the cell cycle, differentiation status, presence of other oncogenic abnormalities and external growth and survival factors as well as the level of DNA damage and p53 induced.
Because p53 induction can lead to apoptosis after DNA damage, acquisition of p53 mutations might promote tumorigenesis, and because many cytotoxic agents act via a p53-dependent pathway, this may lead to resistance to therapy. Thus far about 220 neuroblastomas, the majority of diagnostic pretreatment samples, have been screened for p53 mutations, only five of which were reported to be mutant for p53 (7, 8, 9, 10, 11, 12) . Of these five cases, four were from patients with progressive disease or relapsed neuroblastoma (7 , 10 , 12) . In neuroblastoma cell lines mutant p53 has been reported in four cell lines to date, all of which were obtained from tumor after cytotoxic therapy at progression or relapse (13, 14, 15, 16) .
The aim of this study was to examine two neuroblastoma cell lines derived from the same patient before [SKNBE(1n)] and after cytotoxic therapy [SKNBE(2c)] for p53 mutations and to test p53 function after DNA damage produced by irradiation. It was found that wild-type, functional p53 was present in SKNBE(1n) cells and mutant, nonfunctional p53 in SKNBE(2c) cells. It is possible that acquisition of p53 mutations in neuroblastoma may be a mechanism of chemo- and radioresistance after an initial treatment response.
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Materials and Methods |
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Genetic Analysis.
Cytogenetic analysis, including G banding, of both cell lines was
performed according to standard protocols.
FISH3
of interphase cells was performed using a p53 (17p13) probe
(Oncor) and whole chromosome painting of metaphase cells using a
chromosome 17 probe (Vysis) using methods described previously
(19)
. FISH images were captured and processed on a Vysis
image analysis system.
Sequencing for p53 Mutations.
Total cellular RNA extracted from frozen cell pellets was used to
synthesize full-length p53 cDNA. This was amplified by the PCR, and the
products were gel-purified and sequenced in both directions by
automated dideoxy chain termination sequencing. The sequences obtained
span exons 4 to 10 of the human p53 gene. p53
mutations were confirmed by sequencing the relevant exon from genomic
DNA, by extracting DNA from a frozen cell pellet, and by amplifying the
relevant exons using methods described previously (20)
.
Irradiation.
Exponentially growing cells were treated with 4-Gy gamma irradiation
from a 137Cesium gamma irradiator (Gamma Cell
1000 Elite; Nordion International Inc., Ontario, Canada) at 3.64 Gy/min
and harvested after incubation at 37°C after irradiation at 0, 1, 2,
4, 6, and 24 h for immunocytochemistry and Western blotting.
ICC.
Cell pellets (3 x 107 cells) were
prepared as cytoblocks (Shandon, Pittsburgh, PA), fixed in
formalin, embedded in paraffin, and cut onto adhesive-coated microscope
slides. ICC was performed by the streptavidin-biotin peroxidase
technique after antigen retrieval by microwaving. Endogenous peroxidase
activity was blocked by incubation in 0.5% hydrogen peroxide/methanol
and nonspecific binding of antibody prevented by blocking in normal
rabbit serum diluted 1:10 with Tris-buffered saline (TBS). NCL-p53DO-7
mouse monoclonal antibody was used, which recognizes amino acids 20–25
at the amino terminal of wild-type and mutant p53
(NovoCastra, Newcastle, United Kingdom). Rabbit antimouse biotinylated
secondary antibody (Dako, Glostrup, Denmark) was used at 1:500,
followed by a streptavidin-biotinylated horseradish peroxidase complex
(Dako). Immunostaining was visualized with the chromagen,
3,3-diaminobenzidine tetrachloride (Sigma, St. Louis, MO) as an
insoluble brown product against a hematoxylin nuclear counterstain.
Protein Analysis.
Whole cells were lysed in Laemmli lysis buffer (21)
,
boiled for 10 min, and sonicated, and the protein content was estimated
(Pierce, Rockford, IL). Cell lysate (50 µg) was loaded on to a
4–20% Tris-HCl precast SDS polyacrylamide gel (Bio-Rad, Hercules,
CA) for electrophoresis with a molecular weight marker (Novex,
San Diego, CA). The proteins were transferred to Hybond C
nitrocellulose membrane (Amersham, Amersham, United Kingdom), blocked
in 5% skimmed milk powder in 0.05% Tween 20 in TBS, and probed with
primary antibody. Mouse monoclonal antibodies used were NCL-p53DO-7 at
a dilution of 1:1000, MDM2 (Ab-1; Calbiochem, Cambridge, MA) at 1:100,
WAF1 (Ab1; Calbiochem) at 1:100, NCL-BCL2 (NovoCastra) at 1:100,
NCMII100 (MYCN; gift from Nao Ikegaki, Children’s Hospital of
Philadelphia, Philadelphia, PA) at 1:10, antiactin ascitic fluid
(Sigma) at 1:1000 as a loading control, and polyclonal rabbit BAX
antibody (PharMingen, San Diego, CA) at 1:1000. Peroxidase-conjugated,
affinity-isolated, goat antimouse (Dako) or goat antirabbit (Dako)
secondary antibodies were used at 1:1000 and enhanced chemiluminescence
for protein detection (Amersham). To enhance detection of possible
low-level WAF1 expression in SKNBE(2c) cells, a biotinylated rabbit
antimouse secondary antibody (Dako) was used at 1:1000 followed by
peroxidase-conjugated streptavidin (Dako) at 1:5000, with an enhanced
chemiluminescence molecular weight marker (Amersham). Recombinant human
p53-expressing baculovirus cell lysate and recombinant human
MDM2 protein were used as positive controls. Densitometry was performed
on immunoblots using a Bio Image capture system (Millipore, Bedford,
MA) and CCT camera linked to a Sun-View computer (Sun View
Microsystems, Mountain View, CA).
Cell Cycle Analysis and Apoptosis Measurements.
Cells (2 x 106) were harvested at
0, 6, 24, 48, 72, 96, and 108 h after irradiation and labeled with
propidium iodide, and DNA was analyzed by FACS (Becton Dickinson,
Oxford, United Kingdom). Cell Quest computer software was used to
calculate the proportions of apoptotic cells and cells in different
phases of the cell cycle. Apoptosis was also measured by Hoechst 33258
(8 µg/ml) nuclear staining at 0, 1, 2, 4, 6, 24, 48, 72, 96, and
108 h after irradiation, and the proportion of apoptotic nuclei
was scored from 500 cells counted using a microscope with a UV filter.
Results of three experiments are shown (mean ± SE).
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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. Interphase FISH for SKNBE(1n) cells showed two signals
for p53 (Fig. 1A)
.
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. Consistent with this,
p53 FISH revealed only a single signal in interphase nuclei
(Fig. 1C)
. The karyotypes of these cell lines suggest that they originate from the same tumor, and among the further genetic abnormalities of the SKNBE(2c) cells is monosomy for the segment 17pter to 17q12–21; this mandates to a reduction to hemizygosity for p53.
p53 gene sequencing revealed the SKNBE(1n) cells to be wild-type and the SKNBE(2c) cells mutant. A missense mutation in the SKNBE(2c) cell line in exon 5 was confirmed at codon 135-TGC (cysteine) to TTC (phenylalanine; Ref. 15 ).
p53 Function in the Cell Lines after Irradiation.
SKNBE(1n) cells have low levels of nuclear p53 pretreatment
that increases after DNA damage (Figs. 2a
and 3a)
. The increase in p53 was detectable from 1 h, with a maximum of 18-fold induction 4 h after irradiation
(Figs. 2a
and 3a)
. p53 function as a
transcription factor in this cell line was shown by an up-regulation of
the p53-responsive genes MDM2 and WAF1
(Fig. 2a)
. MDM2 initially decreased after
irradiation and then increased to a maximum of fivefold induction
4 h after irradiation. Similarly, WAF1 increased to a
maximum of fivefold 4–6 h after irradiation (Fig. 2a)
, and
induction of WAF1 was also detectable using ICC (data not
shown). There was no significant change in BAX, BCL2, or
MYCN expression after irradiation (Fig. 2a)
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and 3a)
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In SKNBE(2c) cells there was high baseline accumulation of nuclear
mutant p53 and no further accumulation after 4-Gy
irradiation (Figs. 2a
and 3b)
. There was low
baseline expression of MDM2 but no induction after
irradiation, and a Mr 21,000
WAF1 protein was not inducible despite using a more sensitive method of
detection for WAF1 (Fig. 2a)
. Also, there was no
induction of WAF1 detectable by ICC (data not shown). There
were no significant changes in BAX, BCL2, or MYCN
expression after irradiation (Fig. 2a)
. There was no
evidence of cell cycle arrest after irradiation (data not shown), and
compared with SKNBE(1n) cells, SKNBE(2c) cells underwent low levels of
apoptosis (Figs. 2b
and 3b)
.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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In up to 60% of human cancers, mutation of the p53 tumor
suppressor gene is an important mechanism of chemoresistance
(5)
, but p53 mutations are rare in
neuroblastoma tumors and cell lines (7, 8, 9, 10, 11
, 13)
. In
neuroblastoma cell lines with wild-type p53 reported thus
far, 8 of 11 were obtained before treatment (Table 1)
and an additional two cell lines obtained after chemotherapy were
wild-type for p53 but also were MDM2 amplified,
an additional mechanism of p53 inactivation in other cancers
(5)
. Using a pair of neuroblastoma cell lines obtained
before and after cytotoxic therapy, the current study showed that
wild-type, functional p53 was present in the cell line
established at diagnosis [SKNBE(1n)], and mutant, nonfunctional
p53 was detected in the cell line established when the
patient progressed despite treatment [SKNBE(2c)].
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In the classical Knudson 2 hit model of tumor suppressor gene inactivation, mutation of one allele is followed by loss of the second allele. Mutational analysis of the p53 gene revealed mutant p53 in SKNBE(2c) cells and wild-type p53 in the SKNBE(1n) cells, consistent with this model. The loss of one allele for p53 and a p53 mutation in the remaining allele has been well described in other tumors (6) . The p53 mutation identified in SKNBE(2c) cells was a missense mutation at codon 135 in exon 5 (TGC to TTC), producing an amino acid substitution from cysteine to phenylalanine. Cysteine residues are usually critical to protein structure and function, and this region of the p53 protein is part of the DNA-binding domain, which is essential for the transcriptional transactivational function of p53 (5) . A p53 mutation at codon 135 has been previously reported in a relapsed neuroblastoma (7) , and a cysteine residue mutation at codon 277 in another relapsed neuroblastoma tumor (12) .
The loss of function associated with the p53 mutation in SKNBE(2C) cells was shown by the high level nuclear accumulation of protein in untreated cells and the failure of DNA damage in the form of irradiation to induce up-regulation of the p53-responsive genes (WAF1 and MDM2) and apoptosis. Nuclear, nonfunctional, mutant p53 has been previously reported in a neuroblastoma cell line (24) , but in neuroblastoma cell lines with wild-type p53, its subcellular localization and function are controversial (14 , 24 , 25) . Our own observations are that p53 is predominantly nuclear and functional in wild-type p53 neuroblastoma cell lines4 including SKNBE(1n), as shown in this study by nuclear p53 accumulation after irradiation, up-regulation of WAF1 and MDM2, and induction of apoptosis. Interestingly, despite induction of WAF1 in SKNBE(1n) cells, they showed reduced G1 arrest 24 h after irradiation. This may be related to MYCN amplification because we have recently observed a lack of G1 arrest after irradiation in MYCN-amplified wild-type p53 neuroblastoma cell lines.4
Because SKNBE(2c) cells were isolated after relapse of the patient after cytotoxic treatment with cyclophosphamide, doxorubicin, vincristine, and radiotherapy, it might be predicted that they would be more resistant to these agents than SKNBE(1n) cells. The higher levels of apoptosis after irradiation in SKNBE(1n) cells compared with SKNBE(2c) cells suggest that SKNBE(1n) are more radiosensitive than SKNBE(2c). A previous cytotoxicity study of both of these cell lines reported that SKNBE(2) cells are more resistant to various chemotherapeutic agents than SKNBE(1) (18) . The concentration of drug required to kill 90% SKNBE(2) cells was 9230 times greater than that required for SKNBE(1) cells for doxorubicin and 7, 9, 13, and 30 times greater for etoposide, cisplatin, carboplatin, and melphalan, respectively (18) . Most of these agents act via a p53-dependent pathway, and it is likely that the presence of the p53 mutation in the SKNBE(2c) cells is at least one of the mechanisms of chemo- and radioresistance in this cell line. Whether the p53 mutation occurred as a result of chemotherapy or was present at low levels and was selected for by p53-dependent chemotherapy is unclear. For the different sensitivities reported for doxorubicin and etoposide, there may be other mechanisms of drug resistance involved, such as differences in multidrug-resistant gene and protein expression between the two cell lines, although this would not account for the differences in sensitivities observed for carboplatin, cisplatin, melphalan, and irradiation.
The general absence of p53 mutations at diagnosis in neuroblastoma may explain the initial response of neuroblastoma to therapy, and if p53 mutations subsequently develop, this may be a mechanism of later drug resistance. We are planning to study the frequency of p53 mutations in a series of tumors from which there is both diagnostic pretreatment biopsy material and relapse tumor available. The development of p53 mutations in other relapsed malignancies such as acute lymphoblastic leukemia and Wilm’s tumor has been previously described (6) . The option of considering alternative therapies for relapsed neuroblastoma in which response is either independent of p53 function, such as Taxol (26) , or even enhanced for p53 mutant cells, such as the multinuclear platinum compound BBR3464 (27) , make this an essential area for future study.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by The North of England Children’s
Cancer Research Fund and the United Kingdom Cancer Research Campaign. 
2 To whom requests for reprints should be
addressed, at Cancer Research Unit, The Medical School, Framlington
Place, Newcastle upon Tyne, NE2 4HH, United Kingdom. Phone:
44-191-222-8221 or 44-191-202-3033; Fax: 44-191-222-7556; E-mail: D.A.Tweddle{at}newcastle.ac.uk
3 The abbreviations used are: FISH, fluorescent
in situ hybridization; ICC, immunocytochemistry; FACS,
fluorescence-activated cell sorting; HSR, homogenously staining
region.
4 D. A. Tweddle, A. J. Malcolm, M. Cole,
A. D. J. Pearson, and J. Lunec. p53 cellular localisation and
function in neuroblastoma: evidence for defective G1 arrest
despite WAF1 induction in MYCN amplified cells,
submitted for publication.
Received 5/ 2/00. Accepted 11/ 8/00.
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