|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
NEOPLASIA
From the Department of Haematology, University of
Liverpool, and the CRC Institute for Cancer Studies, University of
Birmingham, United Kingdom.
The well-established association between TP53 mutations
and adverse clinical outcome in a range of human cancers reflects the
importance of p53 protein in regulating tumor-cell growth and survival.
Although it is theoretically possible for p53 dysfunction to arise
through mechanisms that do not involve TP53 mutation, such
a phenomenon has not previously been demonstrated in a sporadic tumor.
Here, we show that p53 dysfunction in B-cell chronic lymphocytic leukemia (CLL) can occur in the absence of TP53 mutation
and that such dysfunction is associated with mutation of the gene
encoding ATM, a kinase implicated in p53 activation. Forty-three
patients with CLL were examined for p53 dysfunction, as detected by
impaired up-regulation of p53 and of the p53-dependent protein
p21CIP1/WAF1 after exposure to ionizing radiation (IR).
Thirty (70%) patients had normal p53 responses and underwent
progressive IR-induced apoptosis. In 13 (30%) patients, p21
up-regulation was markedly impaired, indicating p53 dysfunction. Six
(14%) of these patients with p53 dysfunction had increased baseline
levels of p53, were found to have TP53 mutations, and were
completely resistant to IR-induced apoptosis. In the other 7 (16%)
patients with p53 dysfunction, IR-induced p53 up-regulation and
apoptosis were markedly impaired, but baseline levels of p53 were not
increased, and no TP53 mutations were detected. Each of
these patients was found to have at least one ATM mutation,
and a variable reduction in ATM protein was detected in all 4 patients
examined. This is the first study to provide a direct demonstration
that p53 dysfunction can arise in a sporadic tumor by a mechanism that
does not involve TP53 mutation.
(Blood. 2001;98:814-822) Chronic lymphocytic leukemia (CLL) is characterized
by the accumulation of predominantly nondividing clonal mature B cells in the blood, bone marrow, lymph nodes, and spleen and by a highly variable clinical course.1,2 Some of this variability can be attributed to the tumor-suppressor protein p53. Thus,
TP53 gene defects in CLL are strongly associated with
large-cell transformation,3 resistance to therapy with
purine analogues,4-6 and shortened patient
survival.4-6
By triggering apoptosis or cell-cycle arrest in response to DNA damage,
p53 contributes to the cytotoxic action of many chemotherapeutic agents
and protects the genome from mutagenic insult.7-9 In
quiescent cells, levels of p53 protein are low owing to its short
half-life. After DNA damage, the half-life of p53 becomes
prolonged10 and the protein accumulates in the
nucleus,11 where it regulates the transcription of a
number of genes, including the cyclin-dependent kinase inhibitor
p21WAF1/CIP1,12 the proapoptotic protein
BAX,13 and the antiapoptotic protein BCL-2.14
TP53 mutations typically prolong the half-life of the
protein in the absence of DNA damage and are, therefore, associated with increased basal levels.15 However, even when
activated, mutant p53 protein cannot regulate gene expression because
of its inability to bind to specific DNA sequences.16
Although TP53 mutations occur in only 10% to 15% of
patients with CLL,4-6,17 it is possible that p53
dysfunction occurs in the disease through alternative mechanisms. For
example, 15% to 35% of patients have an extra copy of chromosome
12,18,19 which encodes the p53-inhibitory protein
MDM2.20 Indeed, MDM2 overexpression has been reported in
CLL21,22 and may be associated with trisomy
12.23 However, the effect of such MDM2 overexpression on
p53 activation has not been reported and is not necessarily predictable. Thus, the p53-inhibitory function of MDM2 is itself subject to negative regulation,24 and the basal level of
this protein might therefore have little impact on p53 activation.
Inactivation of the ATM (ataxia telangiectasia mutated) gene
is another potential cause of p53 dysfunction in CLL. ATM is a
high-molecular-weight protein kinase encoded at 11q22-23 that has been
implicated in p53 activation.25 Thus, ATM can associate with and phosphorylate p53 at serine 1526 and is involved
in the dephosphorylation of p53 at serine 37627; both
events are associated with p53 activation. Reduced levels of ATM
protein have been detected in tumor cells from 30% to 40% of patients with CLL,28,29 and ATM mutations have been
demonstrated in a substantial proportion of such cases.29
In addition, as many as 20% of CLL patients have a deletion of
11q22-2319 that, in a proportion of cases, is associated
with a mutation of the remaining ATM
allele.30,31
Although p53 activation in response to ionizing radiation (IR) is
impaired in thymocytes from atm-knockout
mice32,33 and in lymphoblastoid cells from patients with
ataxia-telangiectasia (A-T)34 (most of whom have
bi-allelic truncating ATM mutations and absent ATM
protein25), it is unknown whether the ATM protein defects
that occur in CLL (many of which consist of amino acid substitutions29-31) result in p53 dysfunction. Indeed,
given that the mechanisms involved in p53 activation are complex,
overlapping and, cell-type dependent,35,36 it is unclear
whether ATM is required for p53 activation in CLL cells.
In light of these uncertainties, the aim of the present study was to
determine whether p53 dysfunction could occur in CLL in the absence of
TP53 mutations and, if so, to determine the cause. To do
this, tumor cells were examined for impaired up-regulation of p53 and
of p53-dependent gene products after exposure to IR. Patients with
impaired p53 responses were then examined for TP53 mutations, and those with wild-type p53, for overexpression of MDM2 and
inactivation of ATM. Using this approach, we identified ATM mutations, but not MDM2 overexpression, as a cause of
p53 dysfunction in CLL.
CLL patients
Cell culture
Cytotoxic agents Cells were irradiated at a rate of 3.4 Gy/min using a cesium Cs 137 source. Staurosporine (Calbiochem, Nottingham, United Kingdom) was used at 0.1 µM, a concentration shown in preliminary experiments readily to induce the killing of CLL cells.Detection of p53, p21, BAX, and BCL-2 by Western blot analysis Cultured CLL cells (2 × 106) were lysed for 30 minutes at 4°C in modified RIPA buffer containing 25 mM Tris-HCl (pH 7.6), 1% NP40, 5 mM EDTA, 50 mM sodium chloride, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/mL aprotinin. Lysates were centrifuged at 13 000g for 15 minutes, and the supernatants were mixed with sample buffer, incubated for 15 minutes at 95°C, separated by SDS-PAGE, and transferred to a nylon membrane (Immobilon-P; Millipore, Bedford, MA). After blocking in 5% milk, the membranes were probed with rabbit polyclonal antibodies to p21 (C-19; Santa Cruz Biotechnology, Santa Cruz, CA; 0.5 µg/mL) or to BAX (N-20; Santa Cruz Biotechnology; 0.1 µg/mL), with a mouse monoclonal antibody to BCL-2 (clone 100; Santa Cruz; 0.1 µg/mL) or with a mixture of 2 mouse monoclonal antibodies that bind to both wild-type and mutant p53 (pAb 1801; raised against amino acids 46-55) and pAb 421 (raised against amino acids 371-380), both from Oncogene Science (Cambridge, MA) and used at 0.1 µg/mL.38 After washing, the membranes were reacted with a peroxidase-conjugated goat polyclonal antirabbit or antimouse second-layer antibody (Transduction Laboratories, Lexington, KY). After further washing, specific protein bands were visualized using the enhanced chemiluminescence system (Amersham International PLC, Little Chalfont, Buckinghamshire, United Kingdom).Measurement of ATM by Western blot analysis CLL cells were lysed by sonication in buffer containing 50 mM Tris-HCl (pH 7.5), 9 M urea, and 150 mM -mercaptoethanol. This method of protein extraction was used because it minimizes the problem
of ATM degradation in vitro. Equal amounts of protein extract were
mixed with loading buffer, incubated for 5 minutes at 95°C, separated
by SDS-PAGE, and subjected to Western blot analysis as for p21 and BAX.
Membranes were cut in 2 pieces. The upper part of the membranes
containing high-molecular-weight proteins were probed with a rabbit
polyclonal antibody raised to the N-terminal part of the ATM protein,
AHP397 (Serotec, Oxford, United Kingdom). Results were confirmed by
stripping and reprobing the membranes with a another rabbit polyclonal
anti-ATM antibody, FP8r.29 As a control for protein
loading, the lower part of each membrane was reprobed with a mouse
monoclonal antibody to -actin (AC-74; Sigma). The major bands
corresponding to ATM and -actin were quantified by
densitometry, and the ATM:-actin ratio of each lane was
calculated. To enable comparison between membranes, the ATM: -actin
ratios were normalized with respect to those of the control cells on
the same membrane.
Detection of TP53 mutations Washed CLL cells were lysed in buffer containing 10 mM Tris-HCl (pH 8.0), 0.5 M lithium acetate, 1 mM EDTA, and 2% dodecyl lithium sulfate. Genomic DNA was purified from the lysates by phenol-chloroform extraction and ethanol precipitation.DNA fragments containing TP53 exons and variable amounts of
intron sequence were amplified from approximately 100-ng aliquots of
genomic DNA using Taq polymerase and the supplied buffer (Promega, Southampton, United Kingdom), 3 mM MgCl2, 0.4 mM dNTP, and
20 pmol each primer. For exons 5 to 9, a "touchdown"
protocol39 was used, with the annealing temperature
reduced from 65°C to 55°C over the first 16 of 30 cycles. Exons 4 and 10 were amplified with an annealing temperature of 55°C for 30 cycles. All oligonucleotide primers for polymerase chain reaction (PCR)
were designed from the genomic sequence (accession number, X54156), as
shown in Table 1. Exons 5 and 6 were
coamplified, as were exons 7 and 8, using primers 5/6F with 5/6B and
7/8F with 7/8B, respectively. Exons 4, 9, and 10 were amplified singly
using primers 4F with 4B, 9F with 9B, and 10F with 10B, respectively.
Total RNA was extracted from washed CLL cells essentially as described previously40 but using TRIzol reagent (Gibco BRL, Paisley, United Kingdom). Approximately 5-µg aliquots were reverse transcribed as described40 using Moloney murine leukemia virus reverse transcriptase (Promega) and an oligo(dT)18 primer. Aliquots (1-5 µL each) of the resultant primary-strand cDNA, diluted 1:10 in H2O, were used to amplify the entire coding region (on a 1339-bp fragment) using primers R1F and R11B (Table 1). PCR conditions and reagents were as described above except that 1.5 mM MgCl2 was used and the annealing temperature was reduced from 60°C to 55°C over the first 10 of 30 cycles. PCR products from patient samples and negative controls (no added
DNA/cDNA) were assessed by agarose gel electrophoresis before sequencing. They were prepared for sequencing either by diafiltration using Nanosep 100 K (Pall Filtron, Lichfield, Staffordshire, United Kingdom) centrifugal concentrators (exons 5-6, 7-8, and 9) or after
agarose gel purification and elution using 0.45 µm Nanosep MF (Pall
Filtron) or Genelute (Sigma) spin columns (exons 4 and 10 and reverse
transcription [RT]-PCR fragments). PCR products amplified from
genomic DNA were manually sequenced using a Circumvent kit (New England
Biolabs, Hitchin, Hertfordshire, United Kingdom), and primers were
end-labeled with Detection of ATM mutations CLL patients with p53 defects but without TP53 mutations were subjected to ATM mutation analysis using RT-PCR and either restriction endonuclease fingerprinting (REF), a method described by Liu and Sommer,41 or sequencing of the complete coding region of the ATM gene. For REF analysis, each tumor cDNA was subjected to the amplification of 8 overlapping ATM fragments using primers listed in Table 2. For sequencing of the entire coding region, the amplification of 4 overlapping ATM fragments was initially performed, and then each of the fragments was sequenced with several consecutive primers (Table 2). When feasible, the identified mutation was documented at the tumor DNA level by amplification and sequencing of the corresponding exon from the tumor DNA. In CLL patient 19, ATM mutation analysis was performed from cDNA and from tumor DNA. For this patient, all ATM exons were amplified from the tumor DNA and subsequently fully sequenced using primers previously published.42
Measurement of MDM2 by flow cytometry CLL cells were washed twice in phosphate-buffered saline (PBS), fixed for 60 minutes at room temperature in 1% formaldehyde, washed twice in PBS, permeabilized for 30 minutes at 37°C in 0.5% Tween, washed twice in PBS, and incubated for 30 minutes at room temperature with a mouse monoclonal antibody known to react specifically with MDM2 in formalin-fixed cells (SMP14; Santa Cruz; 5 µg/mL in PBS-BSA-azide) or with a class-specific control antibody. After 2 further washes in PBS, the cells were incubated for 30 minutes at room temperature with a fluorescein isothiocyanate-labeled goat antimouse second-layer antibody (Becton Dickinson; 1:20 dilution in PBS-BSA-azide). After 2 final washes in PBS, staining was quantified by flow cytometry.Measurement of cell killing Cultured cells were gently resuspended and added to an equal volume of PBS containing 10 µg/mL propidium iodide (PI; Sigma). After incubation on ice for 10 minutes, cells were analyzed by flow cytometry. Live cells with intact plasma membranes do not stain with PI. In contrast, dead cells that have lost their membrane integrity take up the fluorochrome and, therefore, exhibit bright red fluorescence.43Detection of apoptosis The mode of cell death was determined by examining CLL cells for DNA laddering and cleavage of poly(ADP-ribose) polymerase (PARP). Low-molecular-weight DNA was extracted as previously described by Forbes et al.44 Briefly, cultured cells (2.5 × 106) were gently washed in PBS and lysed overnight at 4°C in 500 µL buffer containing 5 mM Tris-HCL (pH 7.5), 5 mM EDTA, and 0.5% NP40. Lysates were centrifuged at 13 000g for 20 minutes, and the supernatants were mixed with 100 µL 5 M sodium chloride and 550 µL propan-2-ol. After overnight incubation at 20°C, the precipitated DNA was pelleted and
resuspended in 50 µL buffer containing 10 mM Tris-HCL (pH 7.4) and 1 mM EDTA. Ten-microliter aliquots were mixed with 2.5 µL sample buffer
and subjected to agarose gel (1.5%) electrophoresis. During apoptosis,
internucleosomal DNA cleavage results in the formation of concatamers
of 180 base pairs and a characteristic ladder appearance on
electrophoresis.45
To detect PARP-1 cleavage, cultured cells (2.5 × 106) were gently washed in PBS and lysed in 100 µL sample buffer containing 6 M urea. Lysates were sonicated for 15 seconds and heated at 65°C for 15 minutes before they were subjected to SDS-PAGE and Western blot analysis using an anti-PARP-1 mouse monoclonal antibody (clone A6.4.12; Insight Biotechnology, Wembley, Middlesex, United Kingdom). During apoptosis, intact 116-kd PARP-1 is cleaved by caspases into a characteristic 89-kd C-terminal fragment.46
Detection of p53 dysfunction To screen for p53 dysfunction, CLL cells were examined for an impaired p53 response to IR. To do this, we first tested the effect of radiation on the expression of p53 and of several proteins reported in other cell types to be transcriptionally activated (p21, BAX) or repressed (BCL-2) by p53. A time-course was determined using a single CLL patient with wild-type p53. This patient's cells were known to be highly sensitive to IR-induced killing and were predicted to have a normal p53 response to IR (Figure 1).
As expected, p53 was undetectable at t = 0. However, after irradiation, p53 levels increased, with more protein being detected at 10 hours than at 5 hours or 20 hours. A similar pattern of expression was observed for p21. In contrast, BAX and BCL-2 were readily detectable at t = 0, and levels of these proteins did not change after irradiation. These findings were consistent with the notion that p53 was regulating the expression of p21 (but not BAX or BCL-2), and they indicated that 10 hours would be an appropriate time point for measuring p53 and p21 accumulation in other patients after radiation. Tumor cells from 42 additional CLL patients were next examined for
p53/p21 expression 10 hours after exposure to IR (Figure 2). A normal p53 response (neither p53
nor p21 detectable in nonirradiated cells; both proteins strongly
up-regulated by radiation) was observed in 29 of these 42 patients.
Impaired up-regulation of p21 was detected in 13 patients. In 6 of them
(patients 9, 10, 11, 12, 39, 42), p53 was constitutively increased in
nonirradiated cells and became up-regulated further after
radiation (type A p53 dysfunction). In the other 7 patients with
impaired p21 up-regulation (patients 6, 8, 19, 24, 35, 36, 40), p53 was
not detected in nonirradiated cells, and there was marked impairment of
radiation-induced p53 accumulation (type B p53 dysfunction).
Detection of TP53 mutations All 13 CLL patients and p53 dysfunction were next examined for TP53 mutations by sequencing the entire coding region of the gene (Table 3). A single TP53 mutation was detected in each of the 6 patients with type A p53 dysfunction, and a signal corresponding to the normal sequence was also present in 4 of them (patients 10, 11, 12, 39). In contrast, TP53 mutations were not detected in any of the 7 patients with a type B p53 defect. In each of the 13 patients, the entire coding region could be amplified by RT-PCR. Therefore, the impaired p53 protein expression in the 7 patients with type B dysfunction was unlikely to have resulted from defective transcription caused by mutations in the TP53 promotor region. Taken together, these findings indicate that p53 dysfunction can indeed occur in the absence of TP53 mutation and that this type of dysfunction is associated with normal basal p53 levels and impaired IR-induced p53 protein accumulation and activation.
Measurement of MDM2 To determine whether the type B p53 dysfunction was caused by MDM2 overexpression, levels of this p53 inhibitory protein were measured by indirect immunofluorescence and flow cytometry (Figure 3). Levels of MDM2 were no higher in patients with type B p53 dysfunction than in patients with functionally normal p53 or type A p53 dysfunction. This indicated that type B p53 dysfunction was not caused by MDM2 overexpression.
Measurement of ATM To determine whether the type B p53 dysfunction resulted from ATM inactivation, levels of this protein were measured by Western blot analysis. Because both truncating and missense ATM mutations are associated with a reduction in the amount of full-length ATM protein,29 measurement of the protein can be used to screen for ATM gene defects.Thirty-one CLL tumor samples were examined for ATM expression by
Western blotting using 2 different antibodies. Normal and A-T
lymphoblastoid cells were used as positive and negative controls, respectively (Figure 4). Compared with
the normal lymphoblastoid cells on each membrane, patients with
functionally intact p53 or type A p53 dysfunction (patients 9, 10, 11, 12) had increased amounts of ATM. In contrast, all 4 patients with type
B p53 dysfunction (patients 6, 8, 19, 24) had levels of ATM lower than
those of the normal lymphoblastoid cells. There was, however, some
variation in ATM expression among the 4 patients. Thus, the protein was markedly reduced or undetectable in patients 6, 8, and 24, but in
patient 19, ATM levels were only slightly lower than in those with
normal lymphoblastoid cells. Nonetheless, these findings confirmed that
type B p53 dysfunction was associated with altered levels of ATM
protein.
Detection of ATM mutations We next examined all 7 patients with type B p53 dysfunction for the presence of ATM mutations. To do this, the entire coding region of the ATM gene was subjected to REF and sequencing. This was successfully completed in all 7 patients except patient 19, in whom the 5' end of the transcript could not be amplified despite repeated attempts to do so. Mutations were detected in all 7 patients (Table 4). Two truncating mutations were detected in patient 6, whereas one truncating mutation was detected in patients 24, 35, and 40. In addition, a single silent polymorphism was identified in patients 6 and 24. Two missense mutations were identified in patient 8, whereas one missense mutation was identified in patients 19, 35, and 36. These mutations (except the one in patient 36) all occurred in regions of ATM conserved between mice and humans and were predicted to cause significant changes to amino acids. Furthermore, the sequence alterations in patients 6, 8, 19, and 24 were not present in 40 analyzed meioses. Moreover, none of the alterations in ATM detected in the present study were previously described as polymorphisms in more than 200 A-T patients. Together, these observations strongly suggest that the ATM alterations detected in the present study are true mutations, not polymorphisms.
The difficulty in amplifying the 5' end of ATM from cDNA in
patient 19 was suggestive of major gene disruption. To examine this
possibility, the entire coding region of the ATM gene was sequenced exon by exon from genomic DNA. Three mutations were found.
One (7205A Taken together, these findings confirm the presence of ATM mutations in all 7 patients with CLL with type B p53 dysfunction. Sensitivity to radiation-induced killing We next sought to establish whether ATM mutations in CLL were associated with impairment of p53-dependent cellular functions. Because CLL cells are predominantly nondividing and refractory to mitogenic stimulation,47 we could not study cell-cycle arrest. Therefore, we examined the other main function of p53 the induction of apoptosis. To do this, CLL cells were tested for
their sensitivity to killing by IR, a DNA-damaging agent that induces
p53-dependent apoptosis in mouse thymocytes.48,49
CLL cells with no p53 dysfunction underwent progressive cell death
after exposure to IR (Figure 5). In 2 patients tested, killing was accompanied by extensive DNA laddering and
cleavage of PARP-1 to an 89-kd fragment (data not shown). This
confirmed that the cells were dying by apoptosis. In contrast to CLL
cells with intact p53 responses, cells from all 6 patients with type A
p53 dysfunction and TP53 mutations were completely
radioresistant. This confirmed that the IR-induced killing of CLL cells
was p53 dependent. The 7 patients with CLL with type B p53 dysfunction and ATM mutations were partially resistant to
radiation-induced killing. Thus, little or no cell death was observed
within the first 2 to 3 days after irradiation; thereafter, the cells
underwent a variable amount of delayed killing.
In contrast to the different cytotoxic responses to IR, all 3 groups of CLL cells showed comparable sensitivity to killing by staurosporine and survived equally well under the culture conditions used (Figure 5). This confirmed that the observed differences in IR-induced killing reflected differences in p53 function rather than differences in susceptibility to apoptosis in general.
To our knowledge, the present study provides the first direct demonstration of the notion put forward by Vogelstein and Kinzler in 199250 that p53 dysfunction can occur in sporadic tumors by mechanisms other than TP53 mutation. Although MDM2 overexpression and ATM inactivation have been reported in CLL, the effects of these abnormalities on p53 function in the disease have not previously been studied and were not necessarily predictable. Our demonstration that p53 dysfunction not caused by TP53 mutation is associated with ATM inactivation but not with MDM2 overexpression indicates that p53 activation in CLL cells requires ATM but is largely unaffected by variations in basal levels of MDM2. The implication of ATM as a regulator of p53 function in CLL cells is consistent with the clinical observation that CLL patients with defective expression of ATM have, as do those with TP53 gene defects,4-6 a poor prognosis.28 By showing that patients with CLL with TP53 mutations are completely radioresistant, our data indicate that the radiation-induced killing of CLL cells is p53 dependent, as is the case with mouse thymocytes.48,49 Furthermore, our demonstration that CLL cells with ATM mutations are partially resistant to IR-induced killing is consistent with the idea that ATM regulates p53-mediated apoptosis in CLL cells. The fact that some delayed IR-induced killing occurs in all cells with ATM mutations and impaired p53 up-regulation is entirely consistent with the idea that the wild-type p53 in these cells can be activated by alternative pathways. Thus, it is well known that a delayed p53 response can occur in ATM-deficient cells because of redundancy between ATM and other proteins, such as ATR.51 This may explain why TP53-mutant CLL cells show complete resistance to IR-induced apoptosis, whereas ATM-mutant CLL cells show only partial resistance to such killing. Because p53-mediated apoptosis is thought to underlie the cytotoxic action of many cytotoxic agents,52 our findings implicate ATM mutations as a potential mechanism of drug resistance in CLL and are consistent with the clinical observation that CLL patients with reduced levels of ATM protein respond poorly to therapy.28 Although not every patient with CLL in the present study was examined for TP53 and ATM gene defects, the number of patients with TP53 mutations (14%) was within the expected range of 10% to 15%.4-6,17 Similarly, the frequency of ATM mutations (16%) was close to the 18% found in the only previous study of ATM mutations in which patients were not selected on the basis of having 11q22-23 deletions.29 It is unlikely that the TP53 mutations detected in the present study represent polymorphisms because they have all been previously described in human tumors and because they were all associated with increased baseline levels of p53 protein, impaired radiation-induced up-regulation of p21, and complete resistance to IR-induced killing. Furthermore, the fact that radiation-induced p21 up-regulation and cytotoxicity were completely blocked in patients with p53-mutant CLL that retained a normal copy of the gene is consistent with the idea that mutant p53 can exert a dominant-negative effect over the wild-type protein.53 All 4 CLL patients with ATM mutations who were examined for the ATM gene product had reduced levels of the protein. In agreement with previous reports,29-31 some of these mutations resulted in a frameshift and a premature stop codon, whereas others resulted in an amino acid substitution. The fact that all the alterations within the ATM coding region were associated with reduced ATM protein levels and with impaired radiation-induced up-regulation of p53 and p21 supports the idea that they were true mutations and not polymorphisms. Because cells from A-T patients (most of whom are compound heterozygotes for truncating ATM mutations) but not from A-T carriers display an impaired p53 response to IR, it is likely that the ATM gene defects responsible for impaired p53 activation in CLL cells result from mutation or loss of both alleles. In the 3 CLL patients (patients 6, 8, and 35) in whom more than one ATM mutation was detected, it was not established whether these mutations occurred on the same allele or on different alleles. In one of them (patient 6), however, 2 truncating mutations were associated with complete absence of ATM, and in another (patient 8), 2 missense mutations were associated with marked reduction in the expression of ATM. This is consistent with the mutations being on separate alleles. In each of the other 4 patients with ATM mutations (patients 19, 24, 36, and 40), only one definite ATM mutation was identified within the coding region. In view of the phenotype of these patients with respect to p53 activation, it is likely that a second mutation was present but remained undetected. It was interesting that in patient 19 the ATM gene was not easily amplified from cDNA, and levels of ATM protein were only slightly reduced. Direct sequencing of the gene from genomic DNA revealed 2 splicing errors in addition to the missense mutation already detected. It is difficult to predict the consequences of these mutations at the RNA and protein levels. However, it can be assumed that the first splicing error would lead to the skipping of exon 8 (166 bp), an event predicted to result in the expression of an unstable truncation mutant. The second splicing error should lead to the skipping of exon 12 (372 bp) and an in-frame deletion. This defect might result in some protein expression, explaining why some ATM protein was detected in this patient. Although the protein should be smaller than wild-type ATM, this size difference (320 kd versus 350 kd) is likely to be difficult to detect by Western blotting. It is, however, unclear how the 3 mutations are distributed with respect to the 2 ATM alleles. It is possible that the 2 splicing errors are on separate alleles. If so, the expressed ATM protein could be the in-frame mutant, with or without the missense mutation. Alternatively, if both splicing errors are on the same allele and the missense mutation is on the other, the expressed ATM protein would be a missense mutant. Because the distances between the 3 mutations (8 kb and 80 kb) preclude the separate cloning of the 2 alleles, it remains open to speculation which of these possibilities is correct. Examining the p53/p21 response to IR is clearly an effective way of screening CLL patients for TP53 and ATM mutations. Thus, the fact that these defects were found in all cases with p53 dysfunction indicates that the test has a high level of specificity. Furthermore, the frequencies of TP53 and ATM mutations detected by this method (14% and 16%, respectively) are similar to their reported frequencies, indicating that the test is likely to have a low rate of false-negative results. Screening for ATM deficiency in CLL patients using this functional approach may be more reliable than measuring ATM protein. For example, some tumors with ATM mutations (as in patient 19) might have near-normal levels of ATM protein. Conversely, it is possible that some CLL patients with no ATM mutations might (genuinely or as a result of in vitro protein degradation) express ATM at levels below those of normal lymphoblastoid cells. Therefore, examining CLL patients for p53 dysfunction highly enriches for an ATM-defective subgroup, though the possibility that some ATM mutations might not result in p53 dysfunction cannot be excluded. The main disadvantage of using Western blotting to detect p53 dysfunction is that abnormal subclones might, if present, evade detection. In addition, the method is time consuming and difficult to standardize for routine clinical use. To circumvent these problems, we are exploring ways of detecting impaired p53 activation in individual cells using samples of whole blood. If successful, this should greatly facilitate the recognition of CLL patients with TP53 or ATM mutations, thereby putting this important, but as yet difficult-to-obtain, prognostic information within easy reach of the clinician.
Submitted June 29, 2000; accepted March 28, 2001.
Supported by grants from the Leukaemia Research Fund (United Kingdom), the North West Cancer Research Fund, the Cancer Research Campaign, and the Kay Kendall Leukaemia Fund.
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: Andrew R. Pettitt, Department of Haematology, University of Liverpool, Liverpool L69 3GA, United Kingdom; e-mail: andrew.pettitt{at}rlbuh-tr.nwest.nhs.uk.
1.
Rozman C, Montserrat E.
Chronic lymphocytic leukemia.
N Engl J Med.
1995;333:1052-1057
2.
Caligaris-Capio F, Hamblin TJ.
B-cell chronic lymphocytic leukemia: a bird of a different feather.
J Clin Oncol.
1999;17:399-408 3. Lens D, Dyer M, Garcia-Marco JM, et al. p53 abnormalities in CLL are associated with excess of prolymphocytes and poor prognosis. Br J Haematol. 1997;99:848-857[CrossRef][Medline] [Order article via Infotrieve].
4.
El Rouby S, Thomas A, Costin D, et al.
p53 gene mutation in B-cell chronic lymphocytic leukemia is associated with drug resistance and is independent of MDR1/MDR3 gene expression.
Blood.
1993;82:3452-3459
5.
Wattel E, Preudhomme C, Hecquet B, et al.
p53 mutations are associated with resistance to chemotherapy and short survival in hematologic malignancies.
Blood.
1994;84:3148-3157
6.
Dohner H, Fischer C, Bentz M, et al.
p53 gene deletion predicts for poor survival and non-response to therapy with purine analogues in chronic B-cell leukaemias.
Blood.
1995;85:1580-1589 7. Lane DP. p53, guardian of the genome. Nature. 1992;358:15-16[CrossRef][Medline] [Order article via Infotrieve].
8.
Ko LJ, Prives C.
p53: puzzle and paradigm.
Genes Dev.
1996;10:1054-1072 9. Levine AJ. p53, the cellular gatekeeper for growth and division. Cell. 1997;88:323-331[CrossRef][Medline] [Order article via Infotrieve]. 10. Kastan MB, Onyekwere O, Sidransky D, Vogelstein B, Craig R. Participation of p53 protein in the cellular response to DNA damage. Cancer Res. 1991;51:6301-6311. 11. Fritsche M, Haessler C, Brandner G. Induction of nuclear accumulation of the tumour suppressor protein p53 by DNA-damaging agents. Oncogene. 1993;8:307-318[Medline] [Order article via Infotrieve]. 12. El-Deiry WS, Tokino T, Velculescu VE, et al. WAF1, a potential mediator of p53 tumour suppression. Cell. 1993;75:817-825[CrossRef][Medline] [Order article via Infotrieve]. 13. Miyashita T, Reed J. Tumour suppressor p53 is a direct transcriptional activator of the human bax gene. Cell. 1995;80:293-299[CrossRef][Medline] [Order article via Infotrieve].
14.
Miyashita T, Harigai M, Hanada M, Reed JC.
Identification of a p53-dependent negative response element in the bcl-2 gene.
Cancer Res.
1994;54:3131-3135
15.
Lane DP.
p53 and human cancers.
Br Med Bull.
1994;50:582-599
16.
Prives C.
How loops, 17. Newcombe EW. p53 gene mutations in lymphoid disease and their possible relevance to drug resistance. Leuk Lymphoma. 1995;17:211-221[Medline] [Order article via Infotrieve].
18.
Anastasi J, Le Beau MM, Vardiman JW, et al.
Detection of trisomy 12 in chronic lymphocytic leukemia by fluorescence in situ hybridization to interphase cells: a simple and sensitive method.
Blood.
1992;79:1796-1801
19.
Dohner H, Stilgenbauer S, James MR, et al.
11q deletions identify a new subset of B-cell chronic lymphocytic leukemia characterized by extensive nodal involvement and inferior prognosis.
Blood.
1997;89:2516-2522 20. Oliner JD, Kinzler KW, Meltzer PS, George DL, Vogelstein B. Amplification of a gene encoding a p53-associated protein in human sarcomas. Nature. 1992;358:80-83[CrossRef][Medline] [Order article via Infotrieve].
21.
Watanabe T, Hotta T, Kinoshita T, et al.
The MDM2 oncogene overexpression in chronic lymphocytic leukemia and low-grade lymphoma of B-cell origin.
Blood.
1994;84:3158-3165 22. Bues-Ramos CE, Manshouri T, Haidar MA, Huh YO, Keating MJ, Albitar M. Multiple patterns of MDM-2 deregulation in human leukemias: implications in leukemogenesis and prognosis. Leuk Lymphoma. 1995;17:13-18[Medline] [Order article via Infotrieve]. 23. Hjalmar V, Kimby E, Christensson B. B-cell chronic lymphocytic leukemia: association between the MDM2 and p53 protein expression and trisomy 12 [abstract]. Blood. 1999;94:546. 24. Prives C. Signaling to p53: breaking the MDM2-p53 circuit. Cell. 1998;95:5-8[CrossRef][Medline] [Order article via Infotrieve]. 25. Taylor AMR. What has the cloning of the ATM gene told us about ataxia telangiectasia? Int J Radiol Biol. 1998;73:365-371[CrossRef][Medline] [Order article via Infotrieve]. 26. Khanna KK, Keating KE, Kozlov S, et al. ATM associates with and phosphorylates p53: mapping the region of interaction. Nat Genet. 1998;20:398-400[CrossRef][Medline] [Order article via Infotrieve]. 27. Waterman MJF, Stravridi ES, Waterman JLF, Halazonetis TD. ATM-dependent activation of p53 involves dephosphorylation and association with 14-3-3 proteins. Nat Genet. 1998;19:175-178[CrossRef][Medline] [Order article via Infotrieve].
28.
Starostik P, Manshouri T, O'Brien S, et al.
Deficiency of the ATM protein expression defines an aggressive subgroup of B-cell chronic lymphocytic leukemia.
Cancer Res.
1998;58:4552-4557 29. Stankovic T, Weber P, Stewart G, et al. Inactivation of ataxia telangiectasia mutated gene in B-cell chronic lymphocytic leukaemia. Lancet. 1999;353:26-29[CrossRef][Medline] [Order article via Infotrieve].
30.
Schaffner C, Stilgenbauer S, Rappold GA, Dohner H, Lichter P.
Somatic ATM mutations indicate a pathogenic role of ATM in B-cell chronic lymphocytic leukemia.
Blood.
1999;94:748-753
31.
Bullrich F, Rasio D, Kitada S, et al.
ATM mutations in B-cell chronic lymphocytic leukemia.
Cancer Res.
1999;59:24-27 32. Barlow C, Brown KD, Deng C-X, et al. Atm selectively regulates distinct p53-dependent cell-cycle checkpoint and apoptotic pathways. Nat Genet. 1997;17:453-456[CrossRef][Medline] [Order article via Infotrieve]. 33. Westphal CH, Rowan S, Schmaltz C, Elson A, Fisher DE, Leder P. Atm and p53 cooperate in apoptosis and suppression of tumorigenesis, but not in resistance to acute radiation toxicity. Nat Genet. 1997;16:397-401[CrossRef][Medline] [Order article via Infotrieve]. 34. Khanna KK, Lavin MF. Ionising radiation and UV induction of p53 protein by different pathways in ataxia-telangiectasia cells. Oncogene. 1993;8:3307-3312[Medline] [Order article via Infotrieve]. 35. Prives C, Hall PA. The p53 pathway. J Pathol. 1999;187:112-126[CrossRef][Medline] [Order article via Infotrieve]. 36. Midgeley CA, Owens B, Briscoe CV, Thomas DB, Lane DP, Hall PA. Coupling between gamma irradiation, p53 induction and the apoptotic response depends on cell type in vivo. J Cell Sci. 1995;108:1943-1848. 37. Folkman J, Moscona A. Role of cell shape in growth control. Nature. 1978;273:345-349[CrossRef][Medline] [Order article via Infotrieve].
38.
Zhan Q, Carrier F, Fornace JR.
Induction of cellular p53 activity by DNA-damaging agents and growth arrest.
Mol Cell Biol.
1993;13:4242-4250
39.
Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS.
"Touchdown" PCR to circumvent spurious priming during gene amplification [abstract].
Nucleic Acids Res.
1991;19:4008
40.
Harris RJ, Pettitt AR, Schmutz C, et al.
Granulocyte-macrophage colony-stimulating factor as an autocrine survival factor for mature normal and malignant B lymphocytes.
J Immunol.
2000;164:3887-3893 41. Liu Q, Sommer SS. Restriction endonuclease fingerprinting (REF): a sensitive method for screening in long contiguous segments of DNA. Biol Tech. 1995;18:470-477.
42.
Vorechovsky I, Rasio D, Luo L, et al.
The ATM gene and susceptibility to breast cancer: analysis of 38 breast tumors reveals no evidence for mutation.
Cancer Res.
1996;56:2726-2732 43. Ormerod MG. The study of apoptotic cells by flow cytometry. Leukemia. 1998;12:1013-1025[CrossRef][Medline] [Order article via Infotrieve]. 44. Forbes IJ, Zalewski PD, Giannakis C, Cowled PA. Induction of apoptosis in chronic lymphocytic leukemia cells and its prevenetion by phorbol ester. Exp Cell Res. 1992;198:367-372[CrossRef][Medline] [Order article via Infotrieve]. 45. Wyllie AH, Morris RG. Hormone-induced cell death: purification and properties of thymocytes undergoing apoptosis after glucocorticoid treatment. Am J Pathol. 1982;109:78-87[Abstract].
46.
Kaufmann SH, Desnoyers S, Ottaviano Y, Davidson NE, Poirier GG.
Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis.
Cancer Res.
1993;53:3976-3985 47. Reed JC. Molecular biology of chronic lymphocytic leukaemia. Semin Oncol. 1998;25:11-18[Medline] [Order article via Infotrieve]. 48. Lowe SW, Schmitt EM, Smith SW, Osborne BA, Jacks T. p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature. 1993;362:847-849[CrossRef][Medline] [Order article via Infotrieve]. 49. Clarke AR, Purdie CA, Harrison DJ, et al. Thymocyte apoptosis induced by p53-dependent and independent pathways. Nature. 1993;362:849-852[CrossRef][Medline] [Order article via Infotrieve]. 50. Vogelstein B, Kinzler KW. p53 function and dysfunction. Cell. 1992;70:523-526[CrossRef][Medline] [Order article via Infotrieve]. 51. Zhou B-BS, Elledge SJ. The DNA damage response: putting checkpoints in prospective. Nature. 2000;408:433-439[CrossRef][Medline] [Order article via Infotrieve]. 52. Lowe SW, Ruley HE, Jacks T, et al. p53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell. 1993;74:957-967[CrossRef][Medline] [Order article via Infotrieve]. 53. Milner J, Metcalf EA. Cotranslation of activated mutant p53 with wild-type drives the wild-type p53 protein into the mutant conformation. Cell. 1991;65:765-774[CrossRef][Medline] [Order article via Infotrieve].
© 2001 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  G. G. Johnson, P. D. Sherrington, A. Carter, K. Lin, T. Liloglou, J. K. Field, and A. R. Pettitt A Novel Type of p53 Pathway Dysfunction in Chronic Lymphocytic Leukemia Resulting from Two Interacting Single Nucleotide Polymorphisms within the p21 Gene Cancer Res., June 15, 2009; 69(12): 5210 - 5217. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Zenz, J. Mohr, E. Eldering, A. P. Kater, A. Buhler, D. Kienle, D. Winkler, J. Durig, M. H. J. van Oers, D. Mertens, et al. miR-34a as part of the resistance network in chronic lymphocytic leukemia Blood, April 16, 2009; 113(16): 3801 - 3808. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Rossi, M. Cerri, C. Deambrogi, E. Sozzi, S. Cresta, S. Rasi, L. De Paoli, V. Spina, V. Gattei, D. Capello, et al. The Prognostic Value of TP53 Mutations in Chronic Lymphocytic Leukemia Is Independent of Del17p13: Implications for Overall Survival and Chemorefractoriness Clin. Cancer Res., February 1, 2009; 15(3): 995 - 1004. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Y. H. Hallaert, A. Jaspers, C. J. van Noesel, M. H. J. van Oers, A. P. Kater, and E. Eldering c-Abl kinase inhibitors overcome CD40-mediated drug resistance in CLL: implications for therapeutic targeting of chemoresistant niches Blood, December 15, 2008; 112(13): 5141 - 5149. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. Steele, A. G. Prentice, A. V. Hoffbrand, B. C. Yogashangary, S. M. Hart, E. P. Nacheva, J. D. Howard-Reeves, V. M. Duke, P. D. Kottaridis, K. Cwynarski, et al. p53-mediated apoptosis of CLL cells: evidence for a transcription-independent mechanism Blood, November 1, 2008; 112(9): 3827 - 3834. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Veldurthy, M. Patz, S. Hagist, C. P. Pallasch, C.-M. Wendtner, M. Hallek, and G. Krause The kinase inhibitor dasatinib induces apoptosis in chronic lymphocytic leukemia cells in vitro with preference for a subgroup of patients with unmutated IgVH genes Blood, August 15, 2008; 112(4): 1443 - 1452. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. G. Agrawal, F.-T. Liu, C. Wiseman, S. Shirali, H. Liu, D. Lillington, M.-Q. Du, D. Syndercombe-Court, A. C. Newland, J. G. Gribben, et al. Increased proteasomal degradation of Bax is a common feature of poor prognosis chronic lymphocytic leukemia Blood, March 1, 2008; 111(5): 2790 - 2796. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Saddler, P. Ouillette, L. Kujawski, S. Shangary, M. Talpaz, M. Kaminski, H. Erba, K. Shedden, S. Wang, and S. N. Malek Comprehensive biomarker and genomic analysis identifies p53 status as the major determinant of response to MDM2 inhibitors in chronic lymphocytic leukemia Blood, February 1, 2008; 111(3): 1584 - 1593. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Gine, M. Crespo, A. Muntanola, E. Calpe, M. J. Baptista, N. Villamor, E. Montserrat, and F. Bosch Induction of histone H1.2 cytosolic release in chronic lymphocytic leukemia cells after genotoxic and non-genotoxic treatment Haematologica, January 1, 2008; 93(1): 75 - 82. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Austen, A. Skowronska, C. Baker, J. E. Powell, A. Gardiner, D. Oscier, A. Majid, M. Dyer, R. Siebert, A. M. Taylor, et al. Mutation Status of the Residual ATM Allele Is an Important Determinant of the Cellular Response to Chemotherapy and Survival in Patients With Chronic Lymphocytic Leukemia Containing an 11q Deletion J. Clin. Oncol., December 1, 2007; 25(34): 5448 - 5457. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. N. Damle, S. Temburni, C. Calissano, S. Yancopoulos, T. Banapour, C. Sison, S. L. Allen, K. R. Rai, and N. Chiorazzi CD38 expression labels an activated subset within chronic lymphocytic leukemia clones enriched in proliferating B cells Blood, November 1, 2007; 110(9): 3352 - 3359. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. P. Kater, M. H. J. van Oers, and T. J. Kipps Cellular immune therapy for chronic lymphocytic leukemia Blood, October 15, 2007; 110(8): 2811 - 2818. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Dicker, A. P. Kater, C. E. Prada, T. Fukuda, J. E. Castro, G. Sun, J. Y. Wang, and T. J. Kipps CD154 induces p73 to overcome the resistance to apoptosis of chronic lymphocytic leukemia cells lacking functional p53 Blood, November 15, 2006; 108(10): 3450 - 3457. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Lin, M. A. Glenn, R. J. Harris, A. D. Duckworth, S. Dennett, J. C. Cawley, M. Zuzel, and J. R. Slupsky c-Abl Expression in Chronic Lymphocytic Leukemia Cells: Clinical and Therapeutic Implications. Cancer Res., August 1, 2006; 66(15): 7801 - 7809. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kojima, M. Konopleva, T. McQueen, S. O'Brien, W. Plunkett, and M. Andreeff Mdm2 inhibitor Nutlin-3a induces p53-mediated apoptosis by transcription-dependent and transcription-independent mechanisms and may overcome Atm-mediated resistance to fludarabine in chronic lymphocytic leukemia Blood, August 1, 2006; 108(3): 993 - 1000. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Coll-Mulet, D. Iglesias-Serret, A. F. Santidrian, A. M. Cosialls, M. de Frias, E. Castano, C. Campas, M. Barragan, A. F. de Sevilla, A. Domingo, et al. MDM2 antagonists activate p53 and synergize with genotoxic drugs in B-cell chronic lymphocytic leukemia cells Blood, May 15, 2006; 107(10): 4109 - 4114. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Secchiero, E. Barbarotto, M. Tiribelli, C. Zerbinati, M. G. di Iasio, A. Gonelli, F. Cavazzini, D. Campioni, R. Fanin, A. Cuneo, et al. Functional integrity of the p53-mediated apoptotic pathway induced by the nongenotoxic agent nutlin-3 in B-cell chronic lymphocytic leukemia (B-CLL) Blood, May 15, 2006; 107(10): 4122 - 4129. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Austen, J. E. Powell, A. Alvi, I. Edwards, L. Hooper, J. Starczynski, A. M. R. Taylor, C. Fegan, P. Moss, and T. Stankovic Mutations in the ATM gene lead to impaired overall and treatment-free survival that is independent of IGVH mutation status in patients with B-CLL Blood, November 1, 2005; 106(9): 3175 - 3182. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. L. Kienle, C. Korz, B. Hosch, A. Benner, D. Mertens, A. Habermann, A. Krober, U. Jager, P. Lichter, H. Dohner, et al. Evidence for Distinct Pathomechanisms in Genetic Subgroups of Chronic Lymphocytic Leukemia Revealed by Quantitative Expression Analysis of Cell Cycle, Activation, and Apoptosis-Associated Genes J. Clin. Oncol., June 1, 2005; 23(16): 3780 - 3792. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. Alvi, B. Austen, V. J. Weston, C. Fegan, D. MacCallum, A. Gianella-Borradori, D. P. Lane, M. Hubank, J. E. Powell, W. Wei, et al. A novel CDK inhibitor, CYC202 (R-roscovitine), overcomes the defect in p53-dependent apoptosis in B-CLL by down-regulation of genes involved in transcription regulation and survival Blood, June 1, 2005; 105(11): 4484 - 4491. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Byrd, K. Rai, B. L. Peterson, F. R. Appelbaum, V. A. Morrison, J. E. Kolitz, L. Shepherd, J. D. Hines, C. A. Schiffer, and R. A. Larson Addition of rituximab to fludarabine may prolong progression-free survival and overall survival in patients with previously untreated chronic lymphocytic leukemia: an updated retrospective comparative analysis of CALGB 9712 and CALGB 9011 Blood, January 1, 2005; 105(1): 49 - 53. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. J. Weston, B. Austen, W. Wei, E. Marston, A. Alvi, S. Lawson, P. J. Darbyshire, M. Griffiths, F. Hill, J. R. Mann, et al. Apoptotic resistance to ionizing radiation in pediatric B-precursor acute lymphoblastic leukemia frequently involves increased NF-{kappa}B survival pathway signaling Blood, September 1, 2004; 104(5): 1465 - 1473. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Lozanski, N. A. Heerema, I. W. Flinn, L. Smith, J. Harbison, J. Webb, M. Moran, M. Lucas, T. Lin, M. L. Hackbarth, et al. Alemtuzumab is an effective therapy for chronic lymphocytic leukemia with p53 mutations and deletions Blood, May 1, 2004; 103(9): 3278 - 3281. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. T. Jones, E. Addison, J. M. North, M. W. Lowdell, A. V. Hoffbrand, A. B. Mehta, K. Ganeshaguru, N. I. Folarin, and R. G. Wickremasinghe Geldanamycin and herbimycin A induce apoptotic killing of B chronic lymphocytic leukemia cells and augment the cells' sensitivity to cytotoxic drugs Blood, March 1, 2004; 103(5): 1855 - 1861. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. D. Shanafelt, S. M. Geyer, and N. E. Kay Prognosis at diagnosis: integrating molecular biologic insights into clinical practice for patients with CLL Blood, February 15, 2004; 103(4): 1202 - 1210. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. S. Grewal, J. P. Kahn, M. L. MacMillan, N. K. C. Ramsay, and J. E. Wagner Successful hematopoietic stem cell transplantation for Fanconi anemia from an unaffected HLA-genotype-identical sibling selected using preimplantation genetic diagnosis Blood, February 1, 2004; 103(3): 1147 - 1151. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Stankovic, M. Hubank, D. Cronin, G. S. Stewart, D. Fletcher, C. R. Bignell, A. J. Alvi, B. Austen, V. J. Weston, C. Fegan, et al. Microarray analysis reveals that TP53- and ATM-mutant B-CLLs share a defect in activating proapoptotic responses after DNA damage but are distinguished by major differences in activating prosurvival responses Blood, January 1, 2004; 103(1): 291 - 300. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Starczynski, W. Simmons, J. R. Flavell, P. J. Byrd, G. S. Stewart, H. S. Kullar, A. Groom, J. Crocker, P. A.H. Moss, G. M. Reynolds, et al. Variations in ATM Protein Expression During Normal Lymphoid Differentiation and Among B-Cell-Derived Neoplasias Am. J. Pathol., August 1, 2003; 163(2): 423 - 432. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Lin, S. Manocha, R. J. Harris, Z. Matrai, P. D. Sherrington, and A. R. Pettitt High frequency of p53 dysfunction and low level of VH mutation in chronic lymphocytic leukemia patients using the VH3-21 gene segment Blood, August 1, 2003; 102(3): 1145 - 1146. [Full Text] [PDF]
|
|
|
|
|
|
T. E. Battle, W. G. Wierda, L. Z. Rassenti, D. Zahrieh, D. Neuberg, T. J. Kipps, and D. A. Frank In Vivo Activation of Signal Transducer and Activator of Transcription 1 after CD154 Gene Therapy for Chronic Lymphocytic Leukemia Is Associated with Clinical and Immunologic Response Clin. Cancer Res., June 1, 2003; 9(6): 2166 - 2172. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Vallat, H. Magdelenat, H. Merle-Beral, P. Masdehors, G. Potocki de Montalk, F. Davi, M. Kruhoffer, L. Sabatier, T. F. Orntoft, and J. Delic The resistance of B-CLL cells to DNA damage-induced apoptosis defined by DNA microarrays Blood, June 1, 2003; 101(11): 4598 - 4606. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Oguchi, M. Takagi, R. Tsuchida, Y. Taya, E. Ito, K. Isoyama, E. Ishii, L. Zannini, D. Delia, and S. Mizutani Missense mutation and defective function of ATM in a childhood acute leukemia patient with MLL gene rearrangement Blood, May 1, 2003; 101(9): 3622 - 3627. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Sanchez-Beato, A. Sanchez-Aguilera, and M. A. Piris Cell cycle deregulation in B-cell lymphomas Blood, February 15, 2003; 101(4): 1220 - 1235. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Witkowski, E. Zmuda-Trzebiatowska, J. M. Swiercz, M. Cichorek, H. Ciepluch, K. Lewandowski, E. Bryl, and A. Hellmann Modulation of the activity of calcium-activated neutral proteases (calpains) in chronic lymphocytic leukemia (B-CLL) cells Blood, August 13, 2002; 100(5): 1802 - 1809. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. G. Oscier, A. C. Gardiner, S. J. Mould, S. Glide, Z. A. Davis, R. E. Ibbotson, M. M. Corcoran, R. M. Chapman, P. W. Thomas, J. A. Copplestone, et al. Multivariate analysis of prognostic factors in CLL: clinical stage, IGVH gene mutational status, and loss or mutation of the p53 gene are independent prognostic factors Blood, July 30, 2002; 100(4): 1177 - 1184. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Lin, P. D. Sherrington, M. Dennis, Z. Matrai, J. C. Cawley, and A. R. Pettitt Relationship between p53 dysfunction, CD38 expression, and IgVH mutation in chronic lymphocytic leukemia Blood, July 30, 2002; 100(4): 1404 - 1409. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Gronbak, J. Worm, E. Ralfkiaer, V. Ahrenkiel, P. Hokland, and P. Guldberg ATM mutations are associated with inactivation of the ARF-TP53 tumor suppressor pathway in diffuse large B-cell lymphoma Blood, July 30, 2002; 100(4): 1430 - 1437. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.'i. Saito, A. A. Goodarzi, Y. Higashimoto, Y. Noda, S. P. Lees-Miller, E. Appella, and C. W. Anderson ATM Mediates Phosphorylation at Multiple p53 Sites, Including Ser46, in Response to Ionizing Radiation J. Biol. Chem., April 5, 2002; 277(15): 12491 - 12494. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Stankovic, G. S. Stewart, C. Fegan, P. Biggs, J. Last, P. J. Byrd, R. D. Keenan, P. A. H. Moss, and A. M. R. Taylor Ataxia telangiectasia mutated-deficient B-cell chronic lymphocytic leukemia occurs in pregerminal center cells and results in defective damage response and unrepaired chromosome damage Blood, January 1, 2002; 99(1): 300 - 309. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
[32P] adenosine triphosphate
(Amersham). The RT-PCR purified fragments were sequenced on an ABI
Prism 377 (Applied Biosystems) automated sequencer. For patients 6, 8, 9, 10, 11, 12, 19, and 24, exons 4 to 10 were sequenced from genomic
DNA with the following primers (Table 
G) was previously identified at the RNA level. The other 2 mutations involved the acceptor sites of exons 8 and 12, respectively,
and were identified at the DNA level only (Table 
indicates TP53 mutated (n = 6); 
, ATM mutated (n = 7); and 
, no p53 dysfunction (n = 30).
helices help us to understand p53.
Cell.
1994;78:543-546