Download Hepatitis B Virus Integration Event in Human Chromosome 17p near

(CANCER RESEARCH 51, 49-54, January 1, 1991]
Hepatitis B Virus Integration Event in Human Chromosome 17p near the p53 Gene
Identifies the Region of the Chromosome Commonly Deleted in Virus-positive
Hepatocellular Carcinomas1
Betty L. Slagle,2 Yi-Zhong Zhou, and Janet S. Butel
Division of Molecular Virology, Baylor College of Medicine, Houston, Texas 77030 [B. L. S., J. S. B.j, and Shanghai institute of Biochemistry, Academia Sinica,
Shanghai, China [Y-Z. Z.¡
will depend on the identity and extent of disruption of nearby
genes. Although the activation of cellular oncogenes does not
appear to be a common feature of HBV-associated HCC (3, 5),
the possibility exists that recessive tumor suppressor genes may
play important roles in the development of liver cancer.
Tumor-specific chromosome losses have been detected in
many human tumors by cytogenetics and RFLP studies (1824). The loss of a specific chromosomal fragment in a signifi
cant number of tumors has been thought to result from a
selection process that favored cells that had lost a gene(s) whose
expression was growth inhibitory. RFLP studies on human
liver tumors have identified regions of specific alÃ-eleloss on
chromosomes 1Ip (25), 13q (25), 4 (26,27), and 16q (27); these
observations are consistent with the idea that the loss of genes
on one or more of these chromosomes may be an important
step in the development of some HCCs.
We previously described the cloning and characterization of
a HBV insert from a Chinese HCC. The viral insert mapped to
chromosome 17pl 1.2-12, and cellular sequences were dupli
cated at the site of viral integration (14). In the present study
we used a DNA probe derived from the cellular DNA adjacent
to the previously characterized HBV insert to analyze a set of
19 matched normal liver and HBV-positive hepatoma tissue
samples obtained from the same region of China. Tumorspecific DNA changes were detected in two additional HCCs,
suggesting that the small region of chromosome 17p defined
by the flanking cell DNA probe is frequently altered in hepa
tomas. RFLP studies demonstrated that the loss of one copy of
portions of chromosome 17 occurred often in our sample pop
ulation, including the loss of one alÃ-eleof the p53 gene. As the
p53 gene is known to possess tumor suppressor activity (2830), the functional loss of this gene may be a significant step in
the development of a subset of HBV-positive HCCs.
ABSTRACT
The development of hepatocellular carcinoma (HCC) presumably oc
curs in multiple steps and is influenced by numerous factors. Hepatitis B
virus (HBV) is strongly associated with the development of HCC in
people chronically infected with the virus, but the mechanism of viral
involvement remains unclear. One possibility is that the gross chromo
somal alterations frequently observed in HCC DNA at the site of HBV
integration may alter the expression of important nearby cellular genes.
We previously reported the cloning and characterization of a HBV insert
from a Chinese HCC. The viral insert mapped to chromosome 17pll.212, and cellular sequences were duplicated at the site of viral integration.
In the present study a DNA probe derived from cellular DNA sequences
adjacent to the previously characterized HBV insert was used to analyze
a set of 19 matched normal liver and HBV-positive hepatoma samples
obtained from the same region of China, near Shanghai. Tumor-specific
DNA changes were detected in two additional HCCs, suggesting that the
small region of chromosome 17p defined by the flanking cell DNA probe
is commonly altered in hepatomas. Restriction fragment length polymor
phism studies demonstrated that the loss of one copy of portions of
chromosome 17 occurred in 10 (53%) of the 19 patients. The loss of one
alÃ-eleof the p53 gene (located on chromosome 17pl3) occurred in at
least 6 (60%) of the 10 patients who were heterozygous at the p53 locus.
As the p53 gene is known to possess tumor suppressor activity, the
functional loss of this gene may be a significant step in the development
of a subset of HCCs. High levels of alÃ-eleloss also were detected for
chromosomes 8q (4 of 9; 44%) and 16p (5 of 6; 83%) and may indicate
the presence of additional cellular genes whose functional loss is impor
tant in the development of HCCs.
INTRODUCTION
HCC3 is one of the most common human cancers in Africa
and Southeast Asia (1-5). Although a strong correlation exists
between chronic infection with HBV and the development of
HCC (4, 6, 7), the actual mechanism by which HBV might
contribute to tumor formation remains unknown.
HBV sequences are found integrated in the HMW DNA of
most HCCs obtained from geographic areas with high endemic
HBV chronic infection rates (3, 8). Analyses of integrated HBV
inserts cloned from HCCs have revealed that gross chromo
somal abnormalities (including deletions, duplications, and
translocations) are common at the site of viral integration (917). The functional effects of such cellular DNA changes clearly
MATERIALS
AND METHODS
Tissue Samples and Preparation of Cellular DNA. Hepatoma tissue
and adjacent nontumorous liver tissue were obtained by surgical resec
tion from patients in the Qidong Liver Cancer Institute located near
Shanghai. People's Republic of China. Surgical specimens were placed
immediately into liquid nitrogen and were stored at -80°until trans
ported frozen to the United States. HMW DNA was purified from
tissues as described (8). Characteristics of the 19 members of the sample
population are summarized in Table 1. Patients ranged in age from 26
to 64 years, the majority were male, and all resected tumors contained
integrated HBV sequences.
Probe DNA. Plasmids containing human DNA sequences able to
detect RFLPs were kindly provided by David H. Ledbetter (Institute
for Molecular Genetics, Baylor College of Medicine) or were obtained
from the American Type Culture Collection. Specific information re
garding probe designation, chromosome localization, and restriction
enzyme used is listed in Table 2. The 3.2-kilobase HBV probe was
generated from a cloned head-to-tail dimer of HBV (subtype adw)
Received 9/17/90; accepted 9/28/90.
The costs of publication of this article were defrayed in pan by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by Research Grant CA25215 from the National
Cancer Institute and by The Joseph L. Melnick Professorship of Virology.
2To whom requests for reprints should be addressed, at Division of Molecular
Virology, Baylor College of Medicine, One Baylor Plaza, Houston. TX 77030.
3The abbreviations used are: HCC, hepatocellular carcinoma: HBV, hepatitis
B virus; HMW, high molecular weight: RFLP, restriction fragment length poly
morphism: LOH, loss of heterozygosity.
49
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p5.1 ALLELE LOSS IN HUMAN LIVER CANCER
DNAs to "P-labeled probe DNA was performed as described previously
Table 1 Characteristics of Chinese patients from whom normal liver and
hepatocellular carcinoma tissue samples were obtained
Tumor tissue and nearby nontumorous liver tissue were obtained from patients
in the Qidong Liver Cancer Institute near Shanghai. China. Samples were stored
at —70°
until transported to the United States. The histopathological diagnosis
(14).
Computer Analysis of Autoradiograms. The presence of small
amounts of normal tissue in tumor samples sometimes may contribute
for all tumors was HCC.
a weak autoradiographic signal for one alÃ-elein the tumor sample. In
such instances, densitometer scanning was performed using a Visage
of
60 computer and the Whole-Band analysis program (Biolmage. Ann
integrated
HBV inserts
Arbor, MI) to compare the intensity of both alÃ-elesin normal and
Sample"88-188-288-488-588-688-788-888-1188-1288-1488-1588-1688-1788-1888-1987-2587-2687-2787-28SexMFMMMMMNAMAMMMFMMFMMFAge(years)36315636264464NAN
HBsAg*++NA"NANANANANANANA++NA++NA+++No.
in tumorr5263222322222122122
tumor tissue DNA. In this report, a decrease in intensity of the alÃ-eles
of at least 50% in the tumor sample as compared to the normal DNA
of the same patient was considered to indicate loss of heterozygosity.
RESULTS
Tumor-specific Alterations of Chromosome 17p in HBV-positive HCCs from China. Our laboratory previously described a
HBV insert cloned from a human hepatoma. The viral insert
mapped to chromosome 17pll.2-12 and consisted of an in
verted repeat structure in which adjacent cellular sequences (at
least 3 kilobases) were duplicated. To determine if this region
of chromosome I7p was altered in other liver tumor DNAs, a
set of 19 matched nontumorous and HCC tissues obtained from
China was analyzed using a DNA probe [T7/Bg-E(1.0)] derived
from the cellular DNA that flanked the previously characterized
HBV insert. HMW cellular DNA was digested with EcoR\ and
analyzed by Southern hybridization. A 9-kilobase band of hy
bridization was observed in the normal and tumor DNAs of all
patients (Fig. 1. patients 1, 7, 8, 16, 27, and 28). An additional
4-kilobase band was detected in the normal DNAs of seven
patients (Fig. 1, patients 8, 27, and 28), and this 4-kilobase
band was absent from the tumor DNAs of two patients (Fig. 1,
patients 8 and 27). This result suggests that this small region
of chromosome 17p DNA is altered in at least two other HBVpositive HCCs.
Loss of Chromosome 17 AlÃ-elesin HCC. To determine the
extent and frequency of chromosome 17-specific DNA changes
in our sample population of 19 Chinese patients, we next used
a panel of characterized RFLP DNA probes (Table 2). Genomic
DNAs purified from matched nontumorous and HCC samples
were digested with restriction enzymes appropriate for the
detection of fragment length polymorphisms (Table 2). All 19
patients analyzed were heterozygous at one or more loci on
chromosome 17 (Fig. 2A, patients 88-5N, 88-6N, and 88-7N;
Fig. 28, patient 88-5N; Fig. 2C, patient 88-18N). Twelve
* Each sample was assigned a two-part number. The first two digits indicate
the year in which it was collected: this is followed by a patient number.
'' Information provided by physicians in Shanghai who obtained the tissue
samples. HBsAg. hepatitis B virus surface antigen.
' Hybridization analyses performed on EroRI-digested genomic DNA using
the 3.2-kilobase genomic-length HBV probe.
d NA, data not available.
obtained from William Robinson (31). The retinoblastoma gene probes
were obtained from Rei Takahashi. The 1.0-kilobase fragment of cell
ular DNA that flanked a HBV insert on chromosome I7p (14) was
designated T7/Bg-E(1.0). Probe DNAs were released from plasmid
vectors by restriction enzyme digestion, were isolated by gel electrophoresis in low-melting point agarose gels (SeaPlaque; FMC Corp., Rockland, ME), and were labeled with [12P]dCTP directly within the gel
fragment using a random-primed oligonucleotide labeling reaction
(Boehringer Mannheim Biochemicals, Indianapolis, IN).
Analysis of Matched DNA Samples. Genomic HMW DNAs (10 ^g)
from normal and tumor tissues were digested with the appropriate
restriction enzyme (depending on the probe to be used), and the digested
DNAs were separated by agarose gel electrophoresis. DNA was then
transferred electrophoretically to reusable nylon membranes according
to the specifications of the manufacturer (ZetaProbe; BioRad, Inc.,
Richmond, CA). Hybridization of filters containing bound cellular
Table 2 DNA probes for detection of specific alÃ-eleloss in H'By-associated human HCC
of alÃ-eles
(size
kilobascs)"3(1.7-1.9)>2
in
probeEFD64.1EFD64.2YNM3RB5'RB3'EKMDA2.1YNZ22144D6p5310.5HF12.2YNM67T7/Bg-E(1.0)HHH202HHHI52RMU3THH59MCT6Restriction
or
enzymeTaq\Msp\Msp\,
Chromosome338qI3ql4I3ql4I6pI7pl3.317pl3.317pl3.117pl3I7pl3I7pll.217pl
(65)Nakamura
el al.
(2.6-4.5)>5
(66)Nakamura
et al.
(3.2-3.6)2(2.2-10)2(1.1-19)10(3.0-4.0)>1
EcoRlEcoRl//mdlllRsa\RsaiKsa\Ban\\//mdlllMsp\Rsa\.
(66)Fung el al.
(67)Fung
et al.
(67)Wolff
et al.
(68)Nakamura
et al.
3-2.3)>14
0(1.
(65)Kondoleon
et al.
(1.7-3.3)2(1.5,2.9)*2 (69)McBrideet al.
(70)Schwartz
et al. (33); Kidd et al.
5.3)2(2.1,2.9)2(1.3,3.8)(4.0,
(4.9.
(71)Barkeret al.
(72)Nakamura
et al.
Taq\EcoRlRsa\BamH\Taq\Taq\MsptNo.
(66)Zhou et al.
1.2-1217ql217q24.3-25.117q24.3-25.117q24.3-25.l19Gene
9.0)2(1.9,2.5)2(9.6.
(14)Nakamura
el al.
(66)Nakamura
et al.
10.5)6
(66)Nakamura
et al.
(3.2-3.8)>8
(66)Nakamura
et al.
(3.4-4.0)5(8.5-15)Ref.Nakamura
(66)Niermanet al.
and Maglot (73)
" Certain probes of single-copy DNA can detect multiple alÃ-elesof that DNA because of the presence of variable number tandem repeats associated with that
locus (65).
* In addition to noted alÃ-elesizes, two smaller bands in common to both alÃ-elesmay be detected.
50
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p53 ALLELE LOSS IN HUMAN LIVER CANCER
We used a p53 complementary DNA probe to screen for p53
alÃ-eleloss in our sample population. Ten of the 19 patients
were heterozygous for the two alÃ-elesof p53 (Fig. 3, patients
88-1N, 88-5N, 88-7N, 88-8N, and 88-18N), and a tumorspecific loss of one alÃ-elewas detected in 6 (60%) of the 10
patients (Fig. 3, patients 88- IT, 88-5T, 88-7T, 88-8T, and
88- 1ST; see stars). Quantitative densitometric scanning con
firmed the p53 alÃ-eleloss (>50% reduction in the deleted alÃ-ele;
data not shown), with the faint hybridization signal from the
missing alÃ-elepresumably due to small amounts of contaminat
ing DNA from nontumorous tissue. Although the remaining
p53 alÃ-eleappears normal by digestion with Banll, EcoRI, and
Hinálll (data not shown), the possibility exists that a point
mutation in the remaining alÃ-elemay have affected the func
tional loss of p53 in those tumors.
Loss of Heterozygosity on Chromosomes 3, 8, 16, and 19 in
the HBV-positive HCCs. The finding of chromosome 17p alÃ-ele
loss in 53% of the sample population is similar to the loss rate
reported for other tumors with known involvements of tumor
suppressor genes (18, 36). The absence of cytogenetic data on
noncultured HCC cells makes it difficult to predict the rate of
nonspecific chromosome loss. Similar RFLP studies of human
hepatomas (25, 26), as well as of non-HCC human tumors,
suggest that alÃ-elelosses of 20-25% may be attributed to
genome instability during tumor progression. To assess the
specificity of the 17p LOH in this sample population, we
performed RFLP analyses using probes from four additional
chromosomes. DNA probes specific for chromosomes 3 and 19
revealed <10% LOH (Table 3). In contrast, much higher levels
of alÃ-eleloss were noted for chromosomes 8q (4 of 9 patients;
44% LOH) and 16p (5 of 6 patients; 83% LOH). The LOH for
8q and 16p is higher than the 20-25% expected for random
chromosome loss (25, 26) and may indicate the presence of
8
Fig. 1. Identification of tumor-specific DNA changes in human HCCs using a
probe derived from cellular sequences adjacent to a HBV insert. Paired DNA
samples from nontumorous liver (/V) and matched hepatoma (7") tissues were
digested with EcoRI, and filters were prepared as described in "Materials and
Methods." A 32P-labeled DNA probe (designated 1.0-kilobase flanking DNA
probe) (14). derived from cellular sequences adjacent to a HBV insert, was used
to hybridize with the filter. Representative autoradiograms of the Southern
hybridization are shown. Patients numbered 1, 7, 8. 16, 27. and 28 correspond to
88-1. 88-7, 88-8, 88-16. 87-27. and 87-28, respectively (as described in Table
1). Note the loss of the 4-kilobase band from the tumor (7") DNA of patients 8
and 27.
886
887
N T
N T
2*»
885
N T
-
886
N T
887
N T
1.2
B
88-18
88-19
8726
N T
2 «t »
1,2«
Fig. 2. Tumor-specific loss of heterozygosity for chromosome 17 markers in
HBV-positive hepatoma DNA. Representative autoradiograms of RFLP analyses.
Paired DNA samples from nontumorous liver (N) and matched hepatoma (T)
tissues were digested with the restriction enzyme appropriate for the probe used
(see Table 2). Patients were identified by number according to the assigned code
(see Table 1). The numbers (1 or 2) at the left of each panel identify the two
alÃ-eles,with the number / arbitrarily assigned to the larger of the two alÃ-eles.
Tumor-specific loss is indicated by the arrowheads. Probes used were 144-D6 (A),
YNZ22 (B). and HHH202 (O-
patients were heterozygous at one or more loci on the long arm
of chromosome 17 (17q), and 2 (17%) of the 12 patients
demonstrated a LOH for a 17q marker (Fig. 2C, patient 8818T). In contrast, LOH for markers on 17p occurred much
more frequently. Ten (53%) of the 19 patients had a tumorspecific LOH for 17p markers (Fig. 2A, arrowheads). These
results demonstrate that the loss of one copy of portions of
chromosome 17p is a frequent event in these Chinese HCCs.
Chromosome 17p Alterations Include Loss of the p53 Sup
pressor Gene. The human p53 gene (located on chromosome
17pl3.1) (32-34) has recently been identified as a tumor sup
pressor gene. The loss of normal p53 gene structure has been
associated with the development of several human cancers (35).
1,2
Fig. 3. Tumor-specific loss of p53 heterozygosity in HBV-positive hepatomas.
Representative autoradiograms of RFLP analyses. Paired DNA samples from
nontumorous liver ( \ l and matched hepatoma i /1 tissues were digested with
fiatili and were analyzed by Southern hybridization using a p53 complementary
DNA probe. Patients were identified by number (e.g., 88-5, 88-6) according to
the assigned code (Table 1). The numbers (/ or 2) at the left of each panel identify
the two p53 alÃ-eles,with the number / arbitrarily assigned to the larger of the
two alÃ-eles.Two additional smaller bands present in each lane are in common to
both alÃ-eles(70). Tumor-specific alÃ-eleloss is indicated by the stars. The loss of a
p53 alÃ-ele(>50% reduction in the deleted alÃ-ele)was confirmed by quantitative
densitometer scanning, as described in "Materials and Methods."
Table 3 Loss of heteroiygosity on chromosomes 3, 8, 16, and 19 in HBV-posilive
HCCs from China
Chromosome"
Incidence of LOH*
3«lc
8q
16p
19
1/11(9)"
4/9 (44)
5/6 (83)
0/4 (0)
°Chromosome location of probes used in RFLP analyses.
* Incidence of loss is expressed as the number of patients with tumor-specific
LOH per total number of patients showing constitutional heterozygosity.
c Data for chromosome 3 were derived from two chromosome 3-specific probes,
EFD64.1 and EFD64.2.
d Numbers in parentheses, percentages.
51
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p53 ALLELE LOSS IN HUMAN LIVER CANCER
additional cellular genes whose functional loss is important in
the development of some HCCs.
Pattern of AlÃ-eleLoss in Patients with Chromosome 17 Dele
tions. Chromosome 17 alÃ-eleloss was detected in a total of 10
Chinese hepatoma patients. Analysis of the pattern of alÃ-ele
loss among all 19 patients revealed that the proximal boundary
of common deletion differed among the patients and was not
clearly defined (Fig. 4). Patient 88-28 showed an apparently
unaffected 17pl2 region [detected by the T7/Bg-E(1.0) probe;
Fig. 1] but lost alÃ-elesdistal to that region. Another patient
(88-1) retained both alÃ-elesdetected by YNM67 (map position
17pll.2) but lost alÃ-elesfrom the more distal 17pl3.1 (p53)
and 17pl3.3 (YNZ22). Chromosome 17 markers lost in other
patients included a YNM67 alÃ-ele(Fig. 4, patients 5, 18, and
19). The chromosomal breakpoints differed from patient to
patient, suggesting that the frequent loss of p53 in this sample
population was not due to a fragile site in the chromosome.
Of the 10 patients heterozygous at the p53 locus, 6 (60%)
demonstrated a tumor-specific loss of one alÃ-ele.Two additional
patients who were not heterozygous at the p53 locus (88-6 and
88-17) lost markers both distal and proximal to the p53 locus
and therefore most probably lost a p53 alÃ-ele.Other patients
(88-19 and 88-26) lost 17p markers (detected by probes p 10.5
and YNM67; both proximal to p53) but were homozygous at
all remaining 17p loci. Future quantitative densitometric scan
ning analyses may be able to measure possible p53 alÃ-elelosses
in these four additional patients.
Several patients had LOH for markers on chromosomes 8q
and 16p. All four patients with 8q LOH had concomitant LOH
for markers on chromosome 17 (Fig. 4, patients 8, 17, 19, and
28). In contrast, two cases of chromosome 16p LOH were from
patients with 17p LOH (Fig. 4, patients 6 and 18), and the
remaining three were from patients with an apparently intact
chromosome 17 (Fig. 4, patients 4, 15, and 27). None of the 19
patients displayed LOH for markers on all three chromosomes.
Preliminary analysis of the sample population did not reveal
abnormalities in the retinoblastoma gene (located on chromo
some 13ql4.1; data not shown).
DISCUSSION
The development of HCC probably occurs in multiple steps
and is influenced by multiple factors. HBV is one important
etiological factor in certain parts of the world, such as China
and Africa, where chronic infection with HBV is strongly
associated with the development of HCC. The specific role of
HBV in the development of HCC remains unknown. The
activation of known oncogenes is not a common feature of
HBV-positive HCC (3). A HBV random integration may, in
rare instances, prompt a change in the expression of a critical
regulatory gene by insertional mutagenesis (37-39). However,
it is possible that the functional loss of cellular genes may
contribute change(s) important in the development of liver
cancer. Consistent with the latter, chromosomal alterations are
frequently observed at the site of viral integration in HCC.
RFLP studies on HCCs revealed tumor-specific LOH on
several chromosomes, including 11p [43% LOH (25)], 13q [50%
LOH (25)], 4 [58% LOH (26) and 50% LOH (27)], and 16q
[57% LOH (27)]. In our study of 19 matched liver (nontumor)
and HCC samples from China, a similar level of loss for
chromosome 17p(53% LOH) was detected. The loss of markers
from chromosome 17 was recently demonstrated for other
human cancers, including colon cancer (36), astrocytoma (4042), bladder cancer (43), osteosarcoma (44, 45), lung cancer
(46-49), neurofibrosarcoma (50), and breast cancer (51). One
interpretation of specific alÃ-eleloss in a significant portion of
tumors is that a tumor suppressor gene may be located on that
chromosome. The p53 gene, a known tumor suppressor gene
(28-30, 35), is believed to be the target of the 17p chromosome
deletions in many human cancers (24, 36).
The 17p deletions found in our sample population include
the loss of one alÃ-eleof the p53 gene. Six (60%) of the 10
patients who were heterozygous at the p53 locus had a tumorspecific loss of one alÃ-eleof the p53 gene, and the pattern of
chromosomal changes in an additional four patients was con
sistent with p53 alÃ-eleloss. Thus, it is possible that a total of
10 patients in this sample population of 19 may have lost one
copy of the p53 gene as a result of gross chromosomal alteraPXTXEMT tnJKBBR
1
PROBE•144-OC
YK222•B712-2(17p)(17p)
•p53
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(1»Fig. 4. Pattern of alÃ-eleloss among 19 Chinese HBV-positivc hepatoma patients. Various DNA probes (listed at the left, with chromosome location given in
parentheses) were used for RFLP analyses of matched nontumorous and hepatoma tissue samples. Results of the Southern hybridization assays are listed below the
respective patient identification number. —.homozygosity at that locus in the nontumorous tissue DNA of the patient: O. the presence of both alÃ-eles(heterozygosity)
in tumor DNA: •.tumor-specific loss of one alÃ-ele.
52
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p53 ALLELE LOSS IN HUMAN LIVER CANCER
inactivation may be accomplished either by HBV integration
tions. The normal role of p53 in growth regulation is currently
unclear, and its involvement in cell transformation appears to events or by spontaneous mutations.
be complex (28, 29). In keeping with current models of carcinogenesis, which presume that the inactivation of a tumor
ACKNOWLEDGMENTS
suppressor gene is a two-step process (19, 52), the first event
in a p53-related pathway of transformation is believed to be a
The technical assistance of Ling Tian is gratefully acknowledged.
missense mutation (53) that results in an "activated" p53 known
to contribute to transformation (28-30, 35). The second event
REFERENCES
in p53 loss most probably occurs during the selection of a cell
that has deleted the remaining wild-type p53 alÃ-ele.Such a
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hepatocellular carcinoma: etiologic relationship and clinical implications.
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Princ. Pract. Oncol., /: 1-10, 1987.
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2. Popper. H.. Shafritz. D. A., and Hoofnagle. J. H. Relation of the hepatitis B
virus carrier state to hepatoccllular carcinoma. Hepatology. 7: 764-772.
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1987.
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carcinoma and hepatitis B virus: a prospective study of 22.707 men in Taiwan.
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Lancet. 2: 1129-1133. 1981.
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Hepatitis B Virus Integration Event in Human Chromosome 17p
near the p53 Gene Identifies the Region of the Chromosome
Commonly Deleted in Virus-positive Hepatocellular Carcinomas
Betty L. Slagle, Yi-Zhong Zhou and Janet S. Butel
Cancer Res 1991;51:49-54.
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