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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 3 (August 1), 1999:
pp. 1113-1120
Loss of p73 Gene Expression in Leukemias/Lymphomas Due to
Hypermethylation
By
Seiji Kawano,
Carl W. Miller,
Adrian F. Gombart,
Claus R. Bartram,
Yoshinobu Matsuo,
Hiroya Asou,
Akiko Sakashita,
Jonathan Said,
Eiji Tatsumi, and
H. Phillip Koeffler
From Hematology/Oncology, Cedars-Sinai Medical Center, UCLA School of
Medicine, Los Angeles, CA; Institut fur Humangenetik,
Ruprecht-Karls-Universitat Heidelberg, Heidelberg, Germany; Fujisaki
Cell Center, Hayashibara Biological Laboratories Inc, Okayama, Japan;
the Department of Cancer Genetics, Research Institute for Radiation
Biology and Medicine, Hiroshima University, Hiroshima, Japan; the
Department of Hematology, Saitama Cancer Center, Saitama, Japan; the
Department of Pathology and Laboratory Medicine, UCLA School of
Medicine, Los Angeles, CA; and the International Center for Medical
Research, Kobe University School of Medicine, Kobe, Japan.
 |
ABSTRACT |
The p73 gene, a member of the p53 family, is a new
candidate tumor suppressor gene. To investigate the possibility of
genetic alteration of p73 in leukemia and lymphoma, we examined
55 cell lines and 39 patient samples together with 17 nonhematopoietic cancer cell lines. Gene expression of p73 was detected by
reverse transcriptase-polymerase chain reaction (RT-PCR) in cell lines (5 of 7 pre B/B-acute lymphoblastic leukemia [ALL], 13 of 21 T-ALL/lymphoblastic lymphomas [LBL], 9 of 10 B-non-Hodgkin's
lymphomas [B-NHL], 8 of 9 acute myelogenous leukemias [AML], 2 of 2 T-NHL, 3 of 3 multiple myeloma), and in patient samples (16 of 23 pre
B-ALL, 5 of 8 T-ALL/LBL, 5 of 8 B-NHL). PCR-single-strand conformation
polymorphism (SSCP) of cDNAs showed no mutation in 43 p73-expressing cell lines within the regions that corresponded
to the 5 mutational hotspots of the p53 gene. Neither
homologous deletion nor rearrangement of the p73 gene were
found by Southern blot analysis in any of the cell lines that lack
expression of p73. In contrast to prior published data,
analysis of a polymorphic site showed that the p73 gene was
expressed biallelically in cell lines and normal peripheral blood.
Notably, the p73-negative cell lines were hypermethylated at a
CpG island in the 5' untranslated region of the p73 mRNA, and treatment of these cell lines with 5-azacytidine (5-AC), a demethylation reagent, induced p73 expression. Taken together, we found that a sizable proportion (32%) of ALL/B-NHL cell lines and
primary tumors had negligible or limited expression of the p73
gene associated with hypermethylation of the gene. These findings suggest that silencing of the p73 gene by hypermethylation may contribute to development and/or progression of lymphoid neoplasms.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
MORE THAN HALF of all human cancers
possess alterations of the p53 gene.1-5 Wild-type
p53 is considered to be the guardian of the genome because the protein
markedly increases with DNA injury, resulting in G0/G1 and G2/M
cell-cycle arrest. If DNA injury is too severe, p53 helps to mediate
apoptosis. In contrast, mutant p53 cannot limit cells to a G0/G1 arrest
resulting in unrepaired DNA being bequethed to daughter
cells.5 Recently, a new member of the p53 gene
family has been isolated, known as p73.6 The DNA
binding, transactivation, and oligomerization domains of p73
are similar to those of p53. Overexpression of p73 can induce
the cyclin-dependent kinase inhibitor known as p21waf1 and
cause apoptosis in the p53-negative osteosarcoma cell line, SAOS-2.7 p73 can also activate p53-responsive promotors,
and mutant, but not wild-type, p53 can bind to p73 and inhibit the ability of p73 to both activate p53-responsive promotors and induce apoptosis.8 Taken together, investigators have suggested
that p73 may be a new tumor suppressor.9
Key tumor suppressor genes altered in cancers include p53,
p15INK4B, p16INK4A, and
Rb. These genes can be inactivated by mutation, deletion, and
hypermethylation. Each of these tumor suppressors has been reported to
be altered in lymphoid malignancies.10-13 Furthermore, alterations of the p53, p16INK4A, and
Rb genes have been reported to be associated with an
unfavorable prognosis of acute lymphoblastic leukemias
(ALLs)14-16 and B-non-Hodgkin's lymphomas
(B-NHLs).17-19 To investigate whether p73 is
altered in lymphoid malignancies, we examined cell lines from a variety of hematopoietic malignancies, and because we detected abnormalities in
lymphoid cell lines, patient samples of ALL/B-NHL were also examined. A
sizable number of lymphomas and leukemias had negligible or only
limited expression of the p73 gene. This report implicates hypermethylation as an important mechanism in regulating p73
expression in ALLs/B-NHLs.
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MATERIALS AND METHODS |
Cell lines and primary samples.
Cell lines were cultured in RPMI 1640 or Dulbecco's modified Eagle
medium (DMEM) with 10% fetal bovine serum (Life
Technologies, Gaithersburg, MD). The cell lines listed in
Tables 1 and 2 (21 T-ALL/lymphoblastic lymphoma [LBL], 5 pre B-ALL, 2 B-ALL, 2 T-NHL, 10 B-NHL, 3 multiple myeloma, 9 acute myelogenous leukemia [AML], 3 CML-blastic crisis [CML-BC], and 17 nonhematopoietic cancers) were
either established by our labs20,21 or obtained from
American Type Culture Collection (ATCC; Rockville, MD). Bone marrow and peripheral blood samples were obtained after informed consent from
individuals with lymphoproliferative disorders including T-ALL/LBL (8 samples) and pre B-ALL (23 samples). Mononuclear cells were purified by
density gradient centrifugation from bone marrow and peripheral blood
cells, and immediately frozen in liquid nitrogen. Lymph node tissues
were obtained at the time of surgery from the surgical pathology
division of UCLA Center for the Health Sciences. Tissue blocks
containing malignant lymphoma (confirmed by histologic evaluation of
parallel sections) were snap frozen in liquid nitrogen immediately
after excision, and were used for RNA extraction. In most cases, the
lymphoma had nearly completely effaced the normal architecture of the
lymph node. Lymphomas were classified according to the Revised
European-American classification of lymphoid neoplasms
(REAL),22 and included 2 cases of follicle center lymphoma,
follicular, grade I, 1 case of follicle center lymphoma, follicular,
grade II, 2 cases of small lymphocytic lymphoma, 1 case of
lymphoplasmacytoid lymphoma, and 1 case of mantle cell lymphoma.
Plasmid vectors.
A 2-kbp fragment of the p73 gene was amplified by reverse
transcriptase-polymerase chain reaction (RT-PCR) using Pfu
polymerase (Stratagene, La Jolla, CA) from the cDNA of the
osteosarcoma cell line, U2OS. The primers used for amplification were
5'-GGGGTACCGGCGTGGGGAAGATGGCCCAGT-3' and
5'-GCTCTAGATCAGTGGATCTCGGCCTCCGT-3'. The amplified fragment was gel purified and cloned into the KpnI-XbaI site of
the pBS-M13 vector (Stratagene).
RT-PCR.
Total RNA was extracted using the acid-guanidinium-phenol-chloroform
(AGPC) method,23 and cDNA was synthesized from 5 µg of
total RNA using Moloney murine leukemia virus (M-MLV) Reverse Transcriptase (Life Technologies) in 50 µL of reaction solution according to the manufacturer's instructions. RT-PCR was performed using 10 pmol of each primer, 250 µmol/L of dNTP mix, 10% dimethyl sulfoxide, 2 U of Taq Polymerase (Life Technologies), 1X PCR
buffer (Life Technologies), 150 µmol/L of MgCl2, and 1 µL of cDNA. Primers used for p73 amplification were
5'-GACGGAATTCACCACCATCCT-3' (primer C) and
5'-CCAGGCTCTCTTTCAGCTTCA-3' (primer D). The PCR conditions were 1 cycle of 3 minutes at 95°C, 35 cycles of 30 seconds at 95°C, 40 seconds at 60°C, 40 seconds at 72°C, and 1 cycle
of 3 minutes at 72°C. The quality of cDNA was confirmed by parallel PCR amplification of the GAPDH gene. Primers for GAPDH
were 5'-CCATGGAGAAGGCTGGGG-3' and
5'-CAAAGTTGTCATGGATGACC-3', and the PCR conditions were 1 cycle of 3 minutes at 95°C, 28 cycles of 30 seconds at 95°C, 40 seconds at 58°C, 40 seconds at 72°C, and 1 cycle of 3 minutes at 72°C. PCR products were run on ethidium-bromide (Et-Br)-stained 2% agarose gels and then transferred to Hybond N+ filters (Amersham, Buckingamshire, UK). Membranes were probed with a
[32P] dCTP-labeled 2-kbp XbaI/KpnI
fragment excised and purified from pBS-M13-p73 for p73 and a
0.2-kbp fragment corresponding to the PCR product for GAPDH
using random priming method, and visualized by autoradiography. For
standardization, the photodensity of each p73 band was divided
by that of the corresponding GAPDH band using an Alpha Imager
system (Alpha Innotech, San Leandro, CA), and the expression ratio of
p73/GAPDH for each sample was compared to the ratio of
these two transcripts in HL60. We used HL60 as a standard because the
p73 expression of HL60 was close to that of peripheral blood
mononuclear cells from 4 normal volunteers.
To examine the allelic expression of p73, RT-PCR products were
phenol/chloroform extracted, digested with NlaIII (New England Biolab, Beverly ,MA), run on a 2% gel, and visualized by staining with
Et-Br.
Southern blot analysis.
Genomic DNAs were extracted by standard methods.24 To
detect the deletion of the p73 gene locus, 10 µg of genomic
DNA from each sample was digested with 30 U of EcoRI (Life
Technologies), BamHI (Life Technologies), or HindIII
(Life Technologies) at 37°C overnight. After fractionation on a
0.8% agarose gel, DNA was transferred onto a Hybond N+ membrane. For
probes, a 0.8-kbp EcoRI fragment of pBS-M13-p73, which includes
exons 2 through 6, and a 390-bp fragment of p73 amplified by
PCR with primers C and D (probe CD) , which includes exons 7 through 9, were labeled by random priming with [32P]dCTP. After
hybridization, the membranes were washed stringently and exposed to a
Kodak X-OMAT film (Eastman Kodak, Rochester, NY) for 1 to 5 days.
PCR-single-strand conformation polymorphism (SSCP) and
nucleotide sequencing.
Forty-three cDNA samples from p73-positive cell lines were
screened for mutations by PCR-SSCP analysis. The RT-PCR conditions described above were used to amplify the cDNA in the presence of
[32P]dCTP. This RT-PCR product spans exons 7 through
9, and includes the codons homologous to 5 mutational hotspots (codons
245, 248, 249, 273, and 282) of the p53 gene.1 The
PCR products were denatured and run in a 6% Hydrolink MDE gel (J.T.
Baker, Phillipsburg, NJ) as described by the manufacturer. The PCR
products that showed representative migration patterns on the gel were
purified, and cloned into a pGEM vector (Promega, Madison, WI).
Sequencing was performed using the Original Taq DyeDeoxy Terminator
Cycle Sequencing Kit (Perkin-Elmer, Warrington, UK) and analyzed using
an ABI automated sequencer (Perkin-Elmer).
Methylation analyses.
To assess the methylation status of the p73 gene locus, 0.2 µg of genomic DNA from cell lines or normal PBMC was digested with 20 U of either methylcytosine sensitive enzyme, HpaII (Promega), or its methylation resistant isoschizomer, MspI (Promega), for 3 hours at 37°C. The digests were phenol/chloroform extracted, ethanol precipitated, dried, and resuspended to 10 µL with 1X Tris-EDTA (TE) buffer. One tenth of the solution (20 ng of
DNA) was amplified by PCR using primers:
5'-GGGGACGCAGCGAAACCG-3' and 5'-CTGCAGCCGTCGCAGCC-3', which can amplify the potential
CpG island in exon 1. The PCR conditions were 1 cycle of 3 minutes at
95°C, 30 cycles of 30 seconds at 95°C, 40 seconds at 61°C,
40 seconds at 72°C, and 1 cycle of 3 minutes at 72°C. PCR
products were run on a 3% gel and visualized by staining with Et-Br.
Cell lines that did not express p73 were cultured with
5-azacytidine (5-AC; Sigma, St Louis, MO; a demethylation reagent), to
attempt to induce expression of p73. The U2OS, HL60, CEM,
ALL-Sil, and KOPT-K1 cell lines were cultured either with or without
5-AC at 0.5 to 3 µmol/L for 3 to 5 days.
| |
RESULTS |
Expression of p73 in hematopoietic malignancies.
To investigate the possibility of genetic alteration of p73 in
leukemias/lymphomas, we performed semiquantitative RT-PCR in cell lines
and clinical samples. Each RT-PCR was repeated 2 or 3 times for each
sample with consistent results. Representative results are shown in
Fig 1 and the data are summarized in
Tables 1 and 2. Thirteen of 21 T-ALL/LBL, 5 of 7 pre-B/B-ALL, 9 of 10 B-NHL, and 8 of 9 AML cell
lines were positive for p73 expression by RT-PCR, while
p73 expression was detectable in all nonhematopoietic cancer
cell lines (17 of 17). We also examined the expression of p73
mRNA in fresh ALLs and B-NHLs from patients. Representative results are
shown in Fig 2 and the data are
summarized in Table 3. We found p73-positive
samples in 16 of 23 pre-B-ALL and 5 of 8 T-ALL/LBL, and 5 of 8 B-NHL,
while 13 of 39 samples (33%) of ALLs/B-NHLs had negligible or low
expression of p73 in total. Low expression was arbitrarily set
at less than 1/10 of the level in HL60 cells (<0.1), and an
expression level of less than 1/100 of that in the HL60 cells (<0.01)
was regarded as negligible. Because most samples had some contamination
with normal lymphocytes that express p73 mRNA, this cutoff
selected against false positives. Taken together, a sizable proportion
(32%, 25 of 77) of ALL/B-NHL cell lines and primary tumors had
negligible or limited expression of p73 gene.
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| Fig 1.
p73 expression in pre B/B-ALL, T-ALL/LBL, and
B-NHL cell lines. Autoradiography of RT-PCR products (390 bp) of the
p73 gene is shown in the top panels and GAPDH in the
bottom panels.
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| Fig 2.
p73 gene expression in pre B/B-ALL,
T-ALL/LBL, and B-NHL clinical samples. Autoradiography of
RT-PCR products (390 bp) of the p73 gene is shown in the top
panels and GAPDH in the bottom panels. (A) Lane 1, HL60; lanes
2 through 5, peripheral blood mononuclear cells from 4 volunteers;
lanes 6 through 14, pre B-ALL samples. (B) Lane 1, HL60;
lanes 2 through 9, T-ALL/LBL samples. (C) Lane 1, HL60; lanes 2 through
9, B-NHL samples (lane 2, follicle center lymphoma, follicular, grade
I; lane 3, diffuse large B-cell lymphoma; lane 4, lymphoplasmacytiod
lymphoma; lane 5, small lymphocytic lymphoma; lane 6, small lymphocytic
lymphoma; lane 7, mantle cell lymphoma; lane 8, follicle center
lymphoma, follicular, grade II; lane 9, follicle center lymphoma,
follicular, grade I). Numbers below the lanes represent the ratio of
p73 expression compared with expression in HL60 cells. The
asterisk (*) indicates p73 expression was less than 1/100 of
the level observed in HL60 cells.
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Mutation, deletion, and polymorphism of the p73 gene.
We performed PCR-SSCP analysis with cDNAs to identify possible point
mutations. The primers encompassed the regions corresponding to the 5 mutational hotspots of the p53 gene, which are frequently altered in various cancers, including hematopoietic
malignancies.1 Representative data are shown in
Fig 3A. No mutations in 43 p73-positive cell lines from both hematopoietic and
nonhematopoietic lineages were detected, strongly suggesting that
mutation of the p73 gene might be rare. Two previously
identified polymorphic sites, codon 336 (GCC to GCT) and codon 349 (CAT
to CAC), were identified by sequencing of the RT-PCR
products.25,26 For the p73-nonexpressing cell
lines, we performed Southern blot analysis of the p73 gene locus using 2 different probes, but no alteration of the p73
gene structure was observed (Fig 3B). Taken together, the data suggest that mutation or structural alteration of the p73 gene locus
are rare in hematological malignancies.
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| Fig 3.
Structural analysis of p73 in leukemia and
lymphoma. (A) PCR-SSCP analysis of p73-cDNA from cell lines.
Sequencing of RT-PCR products of representative samples showed that no
mutations were present and the differences in migration of the bands
were derived from the polymorphisms at codons 336 and 349. Arrow (a)
indicates the band appearing when codons 336 and 349 nucleotides are
GCC and CAT, and arrow (b) indicates the band appearing when codons 336 and 349 nucleotides are GCT and CAC, respectively. (B) Southern blot
analysis of the p73 gene from p73 nonexpressing
lymphoid cell lines. (B1) A 0.8-kb EcoRI fragment of
pBS-M13-p73, which covers exons 2 through 6, was used as a probe. Lanes
1 and 7, peripheral blood mononuclear cells; lanes 2 and 8, Hela; lanes
3 and 9, TALL-1; lanes 4 and 10, RPMI8402; lanes 5 and 11, ALL-Sil;
lanes 6 and 12, BALL-1. (B2) Probe CD, which covers exons 7-9, was used
as a probe. Lane 1, Raji; lane 2, HL60; lane 3, HUT78; lane 4, U937;
lane 5, Jurkat; lane 6, SKW3; lane 7, TALL-1; lane 8, CEM; lane 9, ALL-Sil; lane 10, RPMI8402; lane 11, KOPT-K1; lane 12, DS179; lane 13, NALM18; lane 14, BALL-1. Lanes 6, 7, 9, 10, 12, 13, and 14 are
p73-negative cell lines as measured by RT-PCR.
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Because a previous study suggested expression of p73 was
monoallelic,6 we examined our samples for allelic
expression of p73 gene by digesting the RT-PCR products with
NlaIII that identifies the polymorphic site at codon 349 by
cutting at nucleotides CAT but not CAC. In contrast to the prior
report,6 the p73 gene was expressed biallelically
in cell lines and normal PBMC (Fig 4),
although unbalanced expression of the 2 alleles may exist as seen in
the SAOS-2, Daudi, Hela cell lines and peripheral blood mononuclear
cells.
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| Fig 4.
Allelic expression of p73. Differential
expression was detected by NlaIII digestion of RT-PCR products.
The large arrow indicates the uncut band (390 bp). The small arrow (a)
indicates the 358-bp product (NlaIII cuts at codon 223) when
codon 349 is CAC (not cut with NlaIII). The small arrows (b)
and (c) indicate the 277-bp and 89-bp bands, respectively, after
NlaIII cuts at both codon 349 (CAT) and codon 223. C, cut with
NlaIII; U, uncut.
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Methylation status of p73 gene.
We hypothesized that an absence or low level of p73 expression
might be caused by hypermethylation of the CpG island located in exon
1. Hypermethylation and silencing of several tumor suppressor genes,
including p16INK4A, has previously been
noted.27,28 The methylation status of the CG-rich region in
exon 1 of p73 (Fig 5A) was
examined. The genomic DNA was digested in parallel with a
methylcytosine-sensitive and -resistant isoschizomeric enzyme,
HpaII and MspI. The digests were amplified by PCR using
the primer sets that could amplify exon 1. The p73-negative
cell lines (BALL-1, ALL-Sil) were methylated in this region (Fig 5B).
Also, the cell lines with relatively low expression of p73
(Jurkat, Raji, U937, KOPT-K1) were methylated. The
p73-expressing cell lines (U2OS, HL60, BC-1) and normal cells (PBMC1, 2) were not methylated in this region (Fig 5B), consistent with
our hypothesis that methylation of this region was important for the
expression of p73.
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| Fig 5.
(A) Nucleotide sequence of the CpG island in exon 1. Putative methylation sites of exon 1 are illustrated. CG sequences are
bold, and the HpaII/MspI sites are underlined. Numbers
above the nucleotide sequence are based on the cDNA sequence in the
European Molecular Biology Laboratory database under the
accession number Y11416EMBL. (B) Methylation status of CpG island in
exon 1. Digests of 20 ng of genomic DNA with either HpaII or
MspI were amplified by PCR using primers that amplify the
entire exon 1 DNA (77 bp). U, uncut; H, HpaII; M,
MspI.
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To strengthen our hypothesis, we determined if 5-AC (a demethylating
agent) could enhance p73 expression in selected cell lines. The
p73 non- or low-expressing cell lines were cultured with 5-AC
(0.5 to 5 µmol/L) for 3 days. After 3 days of treatment of the
p73-negative cell line, ALL-Sil expressed p73 mRNA at a level comparable to untreated CEM. Furthermore, the p73-low
expressors (KOPT-K1, CEM) increased their p73 expression when
cultured in the presence of 5-AC (Fig 6).
In contrast, levels were unchanged in the U2OS and HL60 cells which
constitutively expressed p73 (Fig 6).
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| Fig 6.
Induction of p73 expression after culture with
5-AC (RT-PCR). U2OS and HL60 were cultured with 5-AC for 3 and 5 days
at 1 and 5 µmol/L, and 0.5 and 3 µmol/L, respectively. CEM,
ALL-Sil, and KOPT-K1 were treated with 5-AC for 3 days at 3 µmol/L.
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DISCUSSION |
Mutations, translocations, and deletions can alter or cause
loss of expression of gene products. The p73 gene is a homolog of p53 and is located at 1p33.2-3, a region that is frequently deleted in neuroblastoma, melanoma, and other cancers.29-32
Forced expression of p73 in p53-negative cells resulted
in their apoptosis.7 Furthermore, some investigators have
claimed that structural abnormalities of p73 occur in some
solid tumors.26,33 Allelic loss of the p73 locus
was observed in 42% (11 of 26 informative cases) of lung
cancers,26 and 5.3% (2 of 38 cases) of prostatic
cancers.33 Because of these observations, p73 is
regarded as a potential tumor suppressor.
To investigate whether alteration of the p73 gene locus occurs
in ALLs/B-NHLs/AMLs, we initially examined the expression of the
p73 gene by RT-PCR using a large variety of hematopoietic cell
lines. Thirty-three percent of ALL/B-NHL cell lines were negative for
p73 gene expression. SSCP analysis of RT-PCR products within
the region that corresponded to the 5 mutational hotspots of the
p53 gene showed no mutation in 43 cell lines (25 hematopoietic) that expressed p73. In contrast, the p53 gene has a
high frequency of mutations in leukemia cell lines that leads to an
overexpression of the aberrant gene.3,34 Furthermore, we
found neither deletions nor translocations of the p73 gene by
Southern blot analysis. Deletions in p53 gene locus occur with
moderate frequency in several human malignancies.4,35 These
results suggest that p53-type mutations rarely occur in the
p73 gene in leukemias/lymphomas.
Nevertheless, a number of hematopoietic cell lines and fresh samples
from individuals had either negligible or very low expression of
p73. Therefore, we looked for a nonmutational mechanism to explain the abberant expression of p73. Imprinting allows the preferential maternal or paternal allele of a gene to be expressed. For
example, retinoblastoma is associated with imprinting of the Rb gene
locus.36 A study suggested that the p73 gene was
imprinted resulting in expression of only 1 allele.6
Quizzically, a second study reported that both alleles were expressed
in lung cancers, but only a single allele was expressed in the matched
normal lung tissues.25 They suggested that the activation
of a silenced allele leading to p73 overexpression may play an
important role in lung cancers. In contrast, a third study found that
25 of 26 normal lung tissue samples expressed both
alleles.26 These same investigators found that the
expression pattern of the 2 alleles varied between various normal
tissues from the same individual. These 3 groups examined the same
polymorphic site in exon 2, but their conclusions were very disparate.
Therefore, we chose to examine another polymorphic site. We found that
both the tumor cell lines and normal peripheral blood cells had
biallelic p73 expression, although several of the cell lines
had an imbalance of expression between the p73 alleles.
The expression of the p16INK4A tumor suppressor
gene is silenced by hypermethylation at the 5' untranslated
region in the course of tumor development in various cancers including
bladder, brain, breast, colon, lung, prostate, and
myelomas.27,37-41 We analyzed the methylation status of the
CpG island (Fig 5A) in the 5' untranslated region of exon 1. Two
p73-negative cell lines (BALL-1, ALL-Sil) had hypermethylation
as detected by digestion with the methylcytosine sensitive enzyme,
HpaII and its isoschizomer MspI, followed by PCR. These
data indicate a correlation between hypomethylation and p73
gene expression. Interestingly, however, 2 cell lines (KOPT-K1, Raji)
expressed p73 but were also hypermethylated in exon 1 of
p73. Possibly other key regions of the gene are hypomethylated in these cell lines. A similar phenomenon has been reported for the
p16INK4A gene in several myeloma cell lines and
fresh breast cancers.42,43 To explore this further, we took
advantage of a well-known demethylating agent. Expression of several
tumor suppressor genes (p16, PTEN) has been induced
after tumor cells were cultured with 5-AC.27,28 This
presumably occurred as a result of hypomethylation of the target genes.
We noted a similar phenomenon when we cultured the p73-nonexpressing cell line (ALL-Sil, Fig 6) with 5-AC.
Taken together, these data suggest that hypermethylation of the CpG
island of p73 might silence expression of the gene in ALL/B-NHL
cell lines and patient samples. Absence or low expression of
p73 may contribute to the development or progression of ALL and
B-NHL.
| |
FOOTNOTES |
Submitted January 7, 1999; accepted April 12, 1999.
Supported in part by National Institutes of Health Grants, the Parker
Hughes Trust, C. and H. Koeffler Fund, and Lymphoma Fundation of
America. H.P.K. is a member of the Jonsson Comprehensive Cancer Center
and holds the endowed Mark Goodson Chair of Oncology Research at
Cedars-Sinai Medical Center/UCLA School of Medicine. S.K. is supported
in part by the Scholarship from Uehara Memorial Foundation.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Seiji Kawano, MD, Hematology/Oncology,
Cedars-Sinai Medical Center, UCLA School of Medicine, Davis Bldg, Room
5065, 8700 Beverly Blvd, Los Angeles, CA 90048; e-mail:
kawanos{at}csmc.edu.
| |
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ABSTRACT
INTRODUCTION