Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2977-2979
CORRESPONDENCE
A Functional Wild-Type p53 Gene Is Expressed in Human Acute
Myeloid Leukemia Cell Lines
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LETTER |
To the Editor:
p53 mutations have been detected in about 10% of all acute myeloid
leukemia (AML) patients, mostly in patients with 17p
monosomy.1-3 The scarcity of p53 mutations in AML could
mean that, in the vast majority of AML patients, loss of p53 protein
function is not required for the development of this disease.
Alternatively, it is possible that inactivation of the p53 growth
regulatory pathway is important and that this can occur either through
disruption of downstream effector molecules or through epigenetic
mechanisms that regulate p53 protein function. It has been suggested,
for example, that inactivation of wild-type p53 protein in AML occurs through a mechanism involving conformational change of the
protein4,5 or through binding to MDM2
protein.6-8 We have examined the functional status of the
wild-type p53 protein expressed in cell lines derived from AML blasts
on the basis of site-specific DNA binding activity, transactivation of
p53-responsive genes, and ability to promote cell cycle arrest in G1 in
response to 
-irradiation.9 The first two properties of
p53 protein are strongly associated with its tumor suppressor
function.10,11
Nucleotide sequence analysis of the entire p53 coding region in four
p53-expressing AML cell lines (OCI/AML-2, -3, -4, and -5)12 showed wild-type sequence. The site-specific DNA
binding activity of p53 protein expressed in OCI/AML-3 and OCI/AML-5
cells was examined using an electrophoretic mobility shift assay
(EMSA). Nuclear protein extracts were prepared from -irradiated or
untreated cells and mixed with a 32P-labeled
double-stranded oligonucleotide containing a p53 binding consensus
sequence, p53CON.13 DNA damage increases the intracellular concentration of p53 protein and is also believed to activate the
latent, sequence-specific DNA binding activity of p53. Whereas little,
if any, DNA binding activity was detected in the nonirradiated extracts, the formation of a p53:DNA complex was evident when extracts
were prepared from irradiated cells (Fig
1). Inclusion of the p53-specific
monoclonal antibody PAb421 in the binding reaction resulted in a
supershifted p53:DNA complex and served to confirm the presence of p53
protein in the protein:DNA complex. DNA binding was not observed when
an extract from the p53-negative cell line Lan1 was used in the EMSA.
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| Fig 1.
DNA binding activity of p53 protein in AML cell lines.
Nuclear extracts prepared from untreated or -irradiated OCI/AML-5
(A) and OCI/AML-3 (B) cells were incubated with a
32P-labeled double-stranded oligonucleotide containing the
p53 consensus sequence (p53CON) with (+) or without ( ) the
p53-specific monoclonal antibody PAb421 and analyzed by EMSA. Lan1
cells, which lack p53 protein, were used as a negative control. The
OCI/AML-5 and Lan1 extracts were prepared 3 hours after -irradiation
with a dose of 6 Gy. The OCI/AML-3 extracts were prepared at the times
indicated after -irradiation with a dose of 2 Gy. The arrow labeled
B points to the p53:DNA complex, and the arrow labeled A points to the
supershifted antibody:p53:DNA complex.
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Activation of p21WAF1 gene transcription after
-irradiation depends on wild-type p53 protein, and the
p21WAF1 gene has been proposed to be a critical downstream
effector in the p53-specific pathway of growth control in mammalian
cells.14-17 Northern blot analysis (Fig
2) indicated that the basal level of
p21WAF1 mRNA was ninefold higher in OCI/AML-5 than in the
mutant p53-expressing human erythroleukemia cell line OCIM2.
Furthermore, 3.5 hours after irradiation with 6 Gy, p21WAF1
mRNA levels increased ninefold in OCI/AML-5 and about threefold in
OCIM2 cells. Irradiated OCI/AML-5 cells contained about 30-fold more
p21WAF1 mRNA than did irradiated OCIM2 cells. No further
increase in p21WAF1 mRNA levels was noted at later times
after irradiation. p21WAF1 induction was also observed in
irradiated OCI/AML-3 and OCI/AML-4 cell lines.18 The mRNA
levels for GADD45 and MDM2, two other genes known to be
transcriptionally regulated by p53 in response to DNA damage, also
increased after -irradiation of AML cell lines (data not shown).
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| Fig 2.
Expression of p21WAF1 mRNA in -irradiated
OCI/AML-5 cells. Samples of total RNA (20 µg) prepared from cells at
different times after exposure to 6 Gy of -irradiation were
fractioned on an agarose-formaldehyde gel, transferred to a nylon
membrane, and hybridized sequentially with 32P-labeled
probes for human p21WAF1 cDNA (A) and 18S ribosomal RNA
(B). OCIM2 cells, which express mutant p53 protein, were used as a
control. Signal intensities were quantitated on a phosphorimager. The
ratio of the p21WAF1 RNA signal to the 18S ribosomal RNA
signal in the OCIM2 sample (0 hours) was arbitrarily set to 1.0 and the
normalized values of p21WAF1 mRNA are shown at the bottom
of (A).
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OCI/AML-3, OCI/AML-5, and OCIM2 cells were irradiated with a dose of 6 Gy and cell proliferation was assessed 16 hours later by propidium
iodide staining and flow cytometry. Both OCI/AML-3 and OCI/AML-5 cells
were blocked in the G1 and G2 phases of the cell cycle with little, if
any, cells present in S phase. In contrast, the mutant p53-expressing
OCIM2 cells accumulated in G2 and showed no evidence of a block in G1
(Fig 3). The failure of OCIM2 cells to
arrest in G1 after -irradiation suggests that the G1 cell cycle
block observed in irradiated OCI/AML-3 and OCI/AML-5 cells is likely to
be dependent on functional p53 protein. Irradiation-induced G1 arrest
was confirmed by dual-parameter flow cytometry after pulse labeling
cells with BrdU and staining for DNA content with propidium iodide and
for BrdU incorporation with a fluorescein isothiocyanate
(FITC)-conjugated antibody for BrdU. OCI/AML-3 and
OCI/AML-5 showed a ninefold and sixfold increase in the G1:S ratio 16 hours after -irradiation (6 Gy), respectively. An increase in the
G1:S ratio provides a good indicator of G1 delay.
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| Fig 3.
Cell cycle changes in AML cells after exposure to
-irradiation (6 Gy). The DNA content was determined by staining the
cells with propidium iodide and the resulting profiles resulting from
propidium iodide fluorescence are shown. For the irradiated cells, the
cell cycle analyses were performed 16 hours after irradiation. OCIM2
cells were used as a control.
|
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Our results indicate that p53 function (DNA binding,
transactivation, and G1 checkpoint) is not lost during the
development of AML or in the establishment of these AML cell
lines. Functional p53 protein has also been demonstrated in
human neuroblastoma,19 non-Hodgkin's
lymphoma,20 and even in certain HPV-positive cancer cell
lines21 that contain wild-type p53 alleles. Hence, loss of
p53 function or inactivation of the p53-dependent growth arrest pathway
is not required for the development of certain malignancies, including
AML.
Trenna Sutcliffe
Loning Fu
Jacinth Abraham
Homayoun Vaziri
Samuel Benchimol
Division of Cellular/Molecular Biology
Ontario Cancer
Institute
Princess Margaret Hospital
Department of Medical
Biophysics
University of Toronto
Toronto, Ontario, Canada
| |
ACKNOWLEDGMENT |
Supported by grants from the Medical Research Council of Canada and the
National Cancer Institute of Canada.
| |
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