Role of p53 in Leukernogenesis of Chronic Myeloid Leukemia
Francesco Lanza,a?’ Sucai
%stitute of Hematology, University of Ferrara, Ferrara, Italy; bLRF Centre for Adult Leukaemia, Royal
Postgraduate Medical School, London, United Kingdom; ‘Department of Molecular Pharmacology, St. Jude
Children’s Research Hospital, Memphis, Tennessee, USA
Key Words. p53 Chronic myeloid leukemia Blast crisis Antisense oligonucleotides Leukemogenesis
Cell cycle
Abstract. This review attempts to provide current
information on the role played by the p53 gene in
normal and leukemic hematopoiesis with particu-
lar emphasis on chronic myeloid leukemia. On the
basis of the currently available data we can argue
that p53 acts as a negative regulator of prolifera-
tion of myeloid mature cells and CD34’ progeni-
tors, and its action is mediated through changes in
cell cycle kinetics, mainly before the S phase. The
p53-dependent pathway is also regulated by sev-
eral proteins, including p16, p21, p27 (cyclin-depen-
dent kinase [CDK] inhibitors), and a few oncogenes
(bcl-2, bax, MDM-2). Although there is some infor-
mation about the changes in the p53 gene seen in
various types of leukemia, the functions and bio-
logical importance of these changes i n the patho-
genesis of leukemia ar e still largely elusive. During
the past several years, accumulated evidence sug-
gests that changes in the p53 gene ar e commonly
associated with blast crisis of chronic myeloid
leukemia (CML) but rarely with chronic phase,
and they are represented by rearrangements, dele-
tions and point mutations. As f o r most of the
tumors, the majority of point mutations occur
between exons 4 and 8 (hot regions). In patients
with CML in blastic crisis the most frequent mecb-
anism of p53 inactivation is complete deletion of
one allele in association with a point mutation in
the remaining allele. As far as the loss of p53 func-
tion in CML patients in blastic crisis is concerned,
we believe that it can play a key role, in combina-
tion with other genetic changes (p210 BCWABL,
Rb gene abnormalities, CDK inhibitors alterations),
in inducing disturbances in proliferation, differ-
entiation and apoptosis of the leukemic clone.
Correspondence: Dr. F. Lanza, Institute of
Hematology, University of Ferrara, St. Anna Hospital,
Via Giovecca n.203, Ferrara 44100, Italy.
Received February 21, 1995; accepted for pub-
lication April 17, 1995. OAlphaMed Press 1066-
5099/95/$5.00/0
STEM CELLS 1995;13:445-452
However, the exact relationship between p210
BCRIABL, the mutant p53, and putative alter-
ations in CDK inhibitors (p21, p16, etc.) in the
pathogenesis of blastic transformation of CML
needs to be clarified. We think that experiments
designed to ascertain whether sustained expres-
sion of a mutant p53 is capable of causing differ-
entiation arrest of Ph-positive hematopoietic cells
in vitro may shed more light on this issue. I t is
also conceivable that a therapeutically-induced
correction of the expression of CDK inhibitors in
p53 mutant CML cells can significantly influence
the cell cycle machinery and possibly suppress
the increased proliferative activity of blastic cells.
Hopefully, highly efficient and targeted wild-type
p53 delivery vectors may, in the future, lead to a
substantial clinical improvement in CML patients
carrying p53 abnormalities a nd undergoing a
blastic transformation of the disease.
Biological Significance of p53 in
Hematopoietic Cells
The p53 protein, a 393 amino acid nuclear
phosphoprotein, is the product of a 20 kb gene
located on the short arm of human chromosome
17 (band: p13), which is one of the more fre-
quent targets for chromosomal aberrations in
human tumors. It contains 11 exons and the
first noncoding exon is separated from the clus-
ter of 10 exons by a large intron measuring 10
kb DNA. The p53 gene codes for a 2.6 kb
mRNA still containing a large noncoding region
that seems to be involved in the stabilization of
the molecule. p53 was first identified as a
tumor-associated antigen in 1979, but it was
not until 1989 that its tumor suppressor potential
was identified. It is now clear that only mutant
forms of p53 have the capability of in vitro
Lanza/Bi
transformation in cooperation with other onco-
genes; the wild type p53, instead, acts as a
tumor suppressor. Comparison of the amino
acid sequences of p53 protein from various
species showed five blocks of highly conserved
regions. Several of these blocks are important in
interactions with other proteins, such as large T
antigen of SV40, E1B of adenovirus and E6 of
the human papillomavirus. They are also the
target regions of frequent mutations in human
tumorigenesis [l-61.
Although the precise role of p53 in cell cycle
control is not yet well understood, it probably
plays a major role in controlling the transition
of cells from GdGl to S phase. Overexpression of
wild-type p53 leads to cell arrest at G , phase
while introduction of mutant p53 has no such
effect. The role of wild-type p53 in mediating
gamma-irradiation-induced GI arrest further sup-
ports the notion that wild-type p53 plays an
important role in mediating cell cycle arrest and
DNA damage response. Interestingly, the phos-
phorylation state of p53 protein is also closely
associated with G,/S phase transition. During
Gc/GIphases, p53 is hypophosphorylated while it
is hyperphosphorylated in S/G2/Mphases [7-141.
Furthermore, it has been shown recently that the
action of p53-mediated GI arrest is dependent
on the induced expression of various cyclin-
dependent kinase (CDK) inhibitors, such as the
p21 WAFl/CIPl and p16. The p21 WAFl/CIPl
gene product is a strong inhibitor of several
CDKs, especially those playing an important role
in G,/S border. Thus, overexpression of p21
WAFl/CIPl leads to inhibition of cell prolifera-
tion and arrest at G, phase [15-181. In addition, it
has been shown that, in response to DNA dam-
aging agents, p53 may arrest cell replication by
activation of the WAFl/CIPl gene, thus inhibit-
ing the activity of the various CDK and cyclin
family of proteins. It has been reported that
increased levels of CDK inhibitors inhibit colony
formation of several myeloid cell lines includ-
ing the K562 chronic myeloid leukemia (CML)
cell line. There is also evidence that the expres-
sion of WAFUCIPl is transcriptionally activated
by wild-type p53 but not by p53 mutants, and
that the levels of this protein are significantly
altered i n myeloid fresh cells from several
leukemic patients [17].
It has been proposed that the wild-type p53
acts as a “molecular policeman” in normal cir-
cumstances, monitoring the integrity of the cell
446
genome. If the DNA integrity is damaged by
either irradiation or other DNA-damaging
reagents, cells will overexpress p53 protein
which leads to cell cycle arrest at GI phase, thus
allowing the cells enough time to repair the
damage, or to begin apoptosis in order to elim-
inate cells with unrepairable genetic defects.
Tumor cells in which p53 is inactivated by
mutation, or by binding to host or viral proteins,
cannot carry out this arrest; they are thus genet-
ically less stable and will accumulate mutations
and chromosomal rearrangements at an
increased rate, leading to rapid selection of
malignant clones [l-6, 19-21].
Apart from conformational changes in p53
proteins caused by point mutations, it has also
been shown that the wild-type p53 protein can
exist in two different conformations: a “native”
wild-type protein, reactive with PAb 1620 mon-
oclonal antibody (mAb) but not PAb 240, which
is associated with antiproliferative and tumor
suppressor activities, and a “mutant” conforma-
tion which reacts with PAb 240 but not with PAb
1620 [22]. It has been postulated that p53 pro-
teins in mutant conformation have no tumor sup-
pressor function. Therefore the changes in p53
protein conformation may reflect the functional
state of wild-type p53 protein during different
stages of cell cycle and proliferation [23-251.
The wild-type p53 protein is present in
minute concentrations in normal cells includ-
ing hematopoietic cells such as neutrophils,
monocytes and lymphocytes [26]. Bone mar-
row CD34- precursors have no detectable p53
protein. However, more than 50% of CD34’
progenitor cells and activated T lymphocytes
express a fair amount of p53 protein [25-281.
In addition, cells from several types of
leukemias contain large amounts of the p53 pro-
tein. The higher concentrations of p53 in
leukemic cells seem to be caused by point muta-
tions or post-translational stabilization [29, 301.
p53 in CML
CML is a hematopoietic malignancy char-
acterized by the Philadelphia (Ph) chromosome
(22q-) formed as a result of a reciprocal translo-
cation between chromosome 9 and 22. At a mol-
ecular level the most important characteristic is
the translocation of the proto-oncogene Abelson
(ABL) from chromosome 9 to the breakpoint
447
cluster region (BCR) on chromosome 22, result-
ing in the formation of BCRIABL hybrid gene
[31, 321. An 8.5 kb mRNA is transcribed from
the hybrid gene and is translated into a fusion
protein (BCR/ABL) of 210 kDa. In a murine
model system, the formation of the chimeric
BCR/ABL is essential for the pathogenesis of a
CML-like disease [33].
CML is a biphasic disease with a fairly
“benign” and relatively manageable chronic
phase. However, in almost all patients blastic
transformation occurred on average three to four
years after the onset of chronic phase, but it seems
not to be caused by further changes in the
chimeric BCWABL gene. The genetic changes
responsible for the transition from chronic phase
to acute phase are poorly defined. However, the
heterogeneity of blast crisis types probably
reflects the variability in molecular and chromo-
somal changes found in this stage of the disease
(two copies of Ph, isochromosome 17q, trisomy 8,
21, 19, etc.). During the past several years, accu-
mulated evidence suggests that changes in the
p53 gene are commonly associated with a blast
crisis of CML but rarely with chronic phase cells
[34-391. Twenty to thirty percent of CML patients
in blastic crisis (BC) show p53 gene alterations,
including gene rearrangements, deletions and
point mutations. The most frequent mechanism
of p53 inactivation in patients with CML in blas-
tic crisis is the complete deletion of one allele in
association with a point mutation in the remaining
allele. The majority of point mutations occur
between exons 4 and 8 (hot regions). Within these
regions, there are four locations where mutations
are found with the highest frequency. These “hot
spots” correspond to amino acid residues 117-
142 (exon 4), 171-181 (exon 5 ) , 234-258 (exon 7)
and 270-286 (exon 8). The half-life of the mutated
p53 protein is usually longer (24-48 h) than that of
its normal counterpart.
Antisense Technology
Although there is some information about
the changes in the p53 gene seen in various
types of leukemia, the functions and biological
importance of these changes in the pathogenesis
of leukemia are still largely elusive. One tech-
nique which permits studies designed to inves-
tigate the role and function of these changes is
antisense technology [25, 40-421. The synthetic
oligonucleotides with sequences complemen-
tary to the target protein coding RNA sequences,
p53 in Chronic Myeloid Leukemia
or sense sequences, are referred to as “anti-
sense” oligonucleotides and appear to inhibit
target gene expression, like natural antisense
RNA, through translation arrest. The antisense
oligonucleotide strategy has been widely used in
studies of cellular functions of oncogenes in
tumorigenesis and the applications have proven
to be successful [25,40-461. The advantages of
using synthetic oligonucleotides as antisense
sequences are as follows: 1) the target mRNA is
less stable in comparison with DNA or protein;
2) the cytoplasmic location of single-stranded
mRNA provides a target readily accessible to
antisense oligonucleotides entering into the cell;
3) it is more efficient than inhibition at the pro-
tein level since one copy of mRNA can theo-
retically give rise to multiple copies of proteins;
4) the duplex formed between DNA/RNA is
more stable than DNA/DNA or RNA/RNA
duplexes and 5 ) the existence of RNase Hybrid
activity which will cleave the RNA strand of
an RNA/DNA duplex, making it even more
attractive because the DNA strand (oligonu-
cleotide) could function like an enzyme, com-
plexing with a target and then freeing itself from
the destroyed target to complex with the next
copy of the target sequence. On the basis of cur-
rent knowledge, it is unlikely that phosphodi-
ester oligodeoxynucleotides can be used
successfully for the in vitro or in vivo treatment
of tumor cells. These compounds are in fact
rapidly hydrolysed by 3‘5’ exonuclease activ-
ity both in plasma and inside the cell. Recently,
the production of nuclease-resistant antisense
oligonucleotides has permitted a broader appli-
cation of this technology. Among these modified
oligomers the most used are phosphorothioate
and the methylphosphonates. The length of the
antisense molecule is crucial in this context, since
it can affect the effectiveness of transportation.
Studies with oligomethylphosphonates of dif-
ferent chain lengths indicated that 8 mers can
be effective in inhibition, but longer oligonu-
cleotides may be more effective. However,
oligonucleotides longer than 21 mers may be
less active, probably because of reduced solu-
bility andor permeability. Therefore the optimal
length of an oligonucleotide should be between
15 and 21 mers.
By using an antisense oligonucleotide
approach we demonstrated that the mutant or trun-
cated p53 proteins expressed by blast cells from
five different CML cell lines (KYO-1, KCL-22,
Lanza/B i
KU812, EM-3, K562) that exhibit abnormali-
ties in p53 gene configuration have no growth-
promoting effect and are not required for cell
survival, proliferation and colony forming units-
granulocyte-macrophage (CFU-GM) produc-
tion [45]. The introduction of p53 antisense
phosphorothioate oligonucleotides specifically
inhibited the translation of the p53 RNA and
the synthesis of the p53 protein. However,
blocking p53 expression had no effect on cell
proliferation, cell viability and colony produc-
tion. As a consequence, we speculated that the
loss of the tumor suppressor function of p53
might not be the only mechanism by which p53
could be involved in the transition from chronic
phase to blast crisis CML [45].
In subsequent studies, we showed that more
than 50% of CD34' hematopoietic progenitor
cells derived from either chronic phase CML
or normal bone marrow from healthy subjects
express p53 protein with different conforma-
tions, suggesting that the function of p53 may be
closely regulated during the cell cycle even in
this primitive cell compartment [25, 271. Also
the mutant conformation-associated PAb 240
antibody was found to be expressed by CD34'
progenitors and activated T lymphocytes of
healthy subjects, allowing us to speculate that
this mAb is not mutant p53 exclusive; in cer-
tain circumstances, the wild-type p53 can also
adopt the "mutant conformation." In addition,
treatment of chronic phase CML cells with p53
antisense oligonucleotides resulted in a signifi-
cantly increased formation of CFU-GM colonies
in 80% of the cases, accompanied by a com-
plete inhibition of p53 expression by CD34'
cells. Furthermore, the expression of different
conformations of p53 in CD34' cell subsets
(CD34+/HLA-DR+,characterized by elevated
levels of PAb 240 and intermediate expression
of PAb 1801; CD34+/HLA-DR-,characterized
by low expression of PAb 240 and high levels of
PAb 1801), as defined by the two p53 mAbs,
was related to the cell cycIe position, thus sup-
porting the concept that the conformational
change of p53 protein may be closely associ-
ated with its functional stage. We therefore
hypothesized that the expression of the wild-
type p53 may be involved in the regulation of
the proliferation pathway of normal and CML
hematopoiesi s.
This finding seems to be relevant in CML
hematopoiesis, since it has been found that the
448
large majority of the leukemic cells carrying
the Ph translocation and the BCR/ABL hybrid
gene are restricted to the CD34'IHLA-DR" cell
subpopulation, which is also characterized by
a higher proliferative activity compared to that
of CD34+/HLA-DR- cell subset.
Our observation was supported by the work
of Zhu et al. [22], who found that the expres-
sion of PAb 240-reactive p53 was closely asso-
ciated with autocrine proliferation of acute
myeloid leukemia (AML) cells, and incubation
of leukemic cells with exogenous growth fac-
tors (GM-CSF) leads to a diminished level of
PAb 1620-reactive p53 while PAb 240-reactive
p53 protein was elevated. Recently they reported
that incubating growth factor-deprived cells
with p53 antisense oligonucleotides suppressed
the expression of PAb 1620' p53 and apopto-
sis [23] without changing the proliferative activ-
ity of the cells. The authors therefore speculated
that the apoptotic process occurring in growth
factor-dependent acute leukemia cells, sec-
ondary to CSFs deprivation, is mediated by
wild-type p53 (PAb 1620') and that conforma-
tional changes of p53 to the PAb 240' immuno-
logic subclass can occur either by mutation or
by the action of autocrine growth factors which
probably prolong leukemic cell survival through
the suppression of apoptosis.
Also in AML patients, it has been reported
that alterations in p53 conformations, rather
than acquisition of point mutations, could be
the principal mechanism underlying the
increased proliferation of myeloid blast cells.
The finding that inhibiting p53 expression
increased CFU-GM colony formation in the
majority of CML samples is consistent with the
observation that blocking synthesis of the
retinoblastoma gene, another tumor suppressor
gene and cell cycle regulator, could release early
hematopoietic cells from quiescence and stimulate
fibroblast cell division and focus formation [47].
In a recent investigation performed by
Mahdi et al., it has been shown that in vitro treat-
ment with p53 and/or retinoblastoma (Rb) anti-
sense oligonucleotides in association with growth
factors (interleukin 3, GM-CSF, interleukin 6,
erythropoietin) induces proliferation of normal
human blood progenitor and stem cells and
increases the number of CFU-GM, burst form-
ing units and CFU-Meg colonies, supporting our
hypothesis that p53 is involved in the control of
distinct intracellular pathways which negatively
449
regulate the growth of hemopoietic progenitor
cells in healthy subjects [48]. In this study, how-
ever, the number of CFU-E colonies was not
affected by p53 antisense treatment, indicating
that p53 may regulate the growth of only a lim-
ited number of progenitor cell subpopulations,
while the remaining, which probably do not
express p53, are not influenced by this negative
regulator. Furthermore, as demonstrated by our
group, the size of the colonies was larger than
in control samples after treatment with p53 anti-
sense oligonucleotides. In a further study, it has
been reported that a selective cytotoxicity to
human leukemic AML cells was produced in
vitro by p53 antisense phosphorothioate oligonu-
cleotides, while the growth of normal bone mar-
row cells was not influenced by this treatment
modality. In addition, the inhibitory effects medi-
ated by p53 antisense oligonucleotides continued
after their removal from the culture medium, sug-
gesting that these compounds can be selectively
toxic to AML blast cells and therefore, thera-
peutically useful in the management of acute
leukemia patients [49].
In line with our previous observation
regarding the influence of p53 antisense
oligonucleotides on the expression of p53 and
their role in regulating proliferation and differ-
entiation of hematopoietic stem cells, we eval-
uated the effects of inhibiting p53 expression
on cell cycle and cell kinetics of either light
density cells or purified CD34’ CML cells [50].
The results showed that treating CML cells with
p53 oligonucleotides in the antisense configu-
ration abrogated p53 expression by either light
density cells or purified (21334’ cells accompa-
nied by a significant increase in the number of
cells positive for the cell cycle specific Ki67
and bromodeoxyuridine mAbs. In addition,
DNA analysis by flow cytometry demonstrated
that the number of cells in quiescent phases of
the cell cycle (Go-G,) was significantly
decreased after exposure of light density cells
with p53 antisense oligonucleotides, while an
increase in the number of cells in either S or
G2-M phases of the cell cycle was observed.
Furthermore, with longer incubation time, the
increase in cell proliferation was higher and the
inhibition of p53 expression was stronger.
Studies on changes in cell kinetics exhibited by
CD34’ cells treated with p53 antisense oligonu-
cleotides also suggested that the conformational
change of p53 protein may be closely linked
p53 in Chronic Myeloid Leukemia
with its functional stage, since the ratio of p53
positivity for the two antibodies differed sig-
nificantly before and after cell incubation with
p53 antisense oligomers. In fact, following p53
antisense oligonucleotide treatment, the num-
ber of cells showing positivity for the p53
immunologic subclass recognized by the PAb
240 mAb was higher than that of 1801 mAb.
We also addressed the functional role of p53
inactivation in the initiation of blastic transfor-
mation of CML by using a retroviral gene trans-
fer technique. We demonstrated that the
introduction of a mutant human p53 cDNA into
hematopoietic progenitor cells from CML
patients in chronic phase (which already contain
p210 BCWABL) could promote growth factor
independent cell proliferation in vitro, but was
unable to generate continuous cell lines [51]. We
postulated that additional genetic aberrations,
other than that involving the p53 gene, might be
required for the complete transformation of CML
into the acute, rapidly fatal stage of the disease.
Recent studies performed with non-
hemopoietic tumor cells showed that the intro-
duction of exogenous wild-type p53 into
nontransformed, wild-type p53-expressing cells
(fibroblasts or adenoma cells) causes no observ-
able effects. In contrast, transduction of normal
p53 into tumor cells that have lost their normal
alleles or bear p53 mutations induces various
effects that range from growth arrest to apoptosis
or differentiation [52]. It will be interesting to
learn what will happen if the wild-type p53 is
introduced into leukemia cells which possess
both wild-type and “mutant” p53 conformations.
The potential clinical application of p53 anti-
sense oligonucleotides in treating acute myeloid
leukemia has been explored. In one report, p53
antisense oligonucleotides were administered
intravenously to patients who showed in vitro
evidence of growth suppression of leukemic cells
but not normal bone marrow by p53 antisense
oligonucleotides [53]. Unfortunately, in this study
there was no data concerning the p53 gene con-
figuration of the leukemic cells. However, the
authors have demonstrated that p53 antisense
oligomers can be administered systemically to
patients without major toxicity, achieving an
adequate bioavailability in the target tissues.
Furthermore, expressed mutant p53 may
represent an attractive option for immunother-
apy of leukemias. This goal can be reached
through the identification of epitopes of p53
LanzdBi
that can be recognized by human cytotoxic T
lymphocytes. The in vitro induction of response
of CD8' T lymphocyte clones can be obtained
using specific peptides of mutant p53 that render
these cells capable of specifically lysing target
cells loaded with tumor specific mutant p53
peptides [54]. However, the clinical relevance of
such an approach in leukemic patients is under
evaluation and more data are needed in order
to confirm these experimental results.
In conclusion, we think that the loss of p53
tumor suppressor function at the time of BC of
CML is not sufficient per se to induce a blastic
phase, but it could act synergistically with other
genetic changes (p210 BCWABL, Rb gene abnor-
mality, p16 deletions, p21 alterations, etc.).
However, the disturbances in cell proliferation,
maturation and apoptosis secondary to p53 gene
abnormalities may be relevant in CML
hematopoiesis and are probably capable of induc-
ing the development of a blastic phenotype in a
considerable number of CML patients. Concerning
the role of p53 in normal and leukemic
hematopoiesis, it can be stated that this tumor sup-
pressor gene probably acts as a negative regulator
of cell proliferation and its action is mediated
through changes in cell cycle kinetics, mainly
before S phase. However, the exact relationship
between p210 BCWABL and the mutant p53 in
the pathogenesis of blastic transformation of CML
needs to be clarified. We believe that experiments
designed to ascertain whether sustained expres-
sion of a mutant p53 is capable of causing differ-
entiation arrest of Ph-positive hematopoietic cells
in vitro may shed more light on this issue. It is
also conceivable that a therapeutically-induced
correction of the expression of CDK inhibitors in
p53 mutant CML cells can significantly influence
the cell cycle machinery and possibly suppress
the increased proliferative activity of blastic cells.
Hopefully, highly efficient and targeted wild-type
$3 delivery vectors may lead in the future to a
substantial clinical improvement in CML patients
carrying p53 abnormalities.
Acknowledgments
We wish to thank Professor John Goldnzan
(LRF Centre for Adult Leukaemia, London,
United Kingdom) for the valuable revision on
the manuscript and for his enthusiastic scientific
support of this project.
450
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