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Shan-Min
Yang, Hong Zhou, Rui-Chuan Chen, Yu-Fang Wang, Fu Chen, Chang-Gong Zhang,
Yun
Zhen, Jiang-Hua Yan, Jin-Hua Su, Cancer Research Center, Xiamen University,
Xiame 361005, Fujian, China
Shan-Min Yang, male, born on 1949-12-17 in Xiamen City, Han
nationality, graduated from Fujian Medical University, director of
Department of Cell Biology, associate professor of Cell Biology,
major in Cell Biology of Tumor, having 50 papers published.
*Supported by the Science Foundation of Department of Publish Health
of Fujian, No.85003-01-11
Correspondence to: Dr. Shan-Min Yang, Cancer Research Center,
Xiamen University, Xiamen 361005, Fujian, China
Telephone:
+86-592-2017309
Received: 1998-11-09
Subject
headings: Liver
neoplasms; carcinoma, hepatocellular; p53 gene; mutation; HHC4;
HHC15; Tumor cell, cultured
Yang SM, Zhou H, Chen RC, Wang YF, Chen F, Zhang CG, Zhen Y, Yan JH,
Su JH. Sequencing of p53 mutation in established human
hepatocellular carcinoma cell line of HHC4 and HHC15 in nude
mice.World J Gastroenterol, 1998;4(6):506-510
Abstract
AIM: To set up cell lines of human hepatocellular carcinoma
in nude mice for the research of cell biology and gene therapy.
METHODS: Xenotransplantation of human hepatoma into nude mice
was carried out and the growth rate, histopathology and immunology
of the nude mice were studied. The DNA from xenografts were analyzed
by HBV gen and PCR amplification of a fragment of p53 gene exon 7,
which were identified by dot blot hybridization,restriction
fragments length polymorphism and DNA sequencing.
RESULTS: HHC4 and HHC15 cell lines could be successively
transplanted in nude mice and the population doubling time was 7 and
5 days respectively. These strains retained the original
characteristics of histopathology, secreting AFP and heteroploid
karyotypes in human hepatocellular carcinoma. The fragment of HBV
gene was detected in the genomic DNA of both hHCC4 and hHCC15,
however only HHC4 secreted HBsAg. The mutation at 250 code (C→A)
and 249 code (G→T)
were detected respectively in the genomic DNA of HHC4 and HHC15.
CONCLUSION: The two cell lines are useful material for the
studying of cell biology and gene therapy in human hepatocellular
carcinoma and provide molecular biological trace of relationship
between high mortality of hepatoma and AFB1 severe pollution of the
daily common foods in this district.
INTRODUCTION
Although the etiopathology of human hepatocellular carcinoma (hHCC)
is still unknown, a lot of evidence strongly suggested that
infection of HBV and contamination of aflatoxin B1 (AFB1)
were the inducing fuctos of the carcinogenesis of hHCC. Xenografts
of human tumorous tissue in nude mice usually retain their original
morphology, antigen, karyotype and function. These models can be
used for several purposes, including assessment of the etiopathology,
cell biology, sensitivity of chemotherapy, genetherapy, and so on.
HHC4 and HHC15 cell lines were established from the patients of HHC
in Tong′an
district of Xiamen where there was high adjust death rates of HHC
(44.75 per one hundred thousand population from 1987 to 1989) which
was the secondary mortality of hHCC in China. In this district, a
lot of evidence has been shown that there was severe contamination
of AFB1 in daily common foods, such as the oil of peanut
(92.9%) and fermented soy beans (44.4%)［1］.
Also high epidemic infection of HBV was presented in the population
in this country (17.6%)［2］.
Because the relationship between molecular biological changes and
etiopathology of HHC in this district is still absent hHCC4 and
hHCC15 cell lines in nude mice were studied by cell biology and
molecular biology.
MATERIAL AND METHODS
Animals
Male and female nude mice, about 4 to 6 weeks old, with BALB/C
genetic background were provided by Medical Experimental Animal lab.
in Cancer Research Center, Xiamen University, where the mice were
bred and maintained in vinyl box isolated under specific pathogene
free condition. The sterilized food pellet (B k Co. Ltd, Shanghai)
and tap water were given ad libitum.
Xenotransplantation
Tumor tissues of patients, who were operated by partly hepatotomy in
the Min-Hai Hospital, Tong′an,
were dissected from the primary site in the liver and aseptically
minced and placed in cooled culture medium. Several tumor tissue
fragments about 2mm in diameter were transplanted, with trocar, into
the subcutaneous tissue of the back of 6 mice within 3 hours after
surgical removal of the tumor. Afterwards, a piece of tumor tissue
from HHC15 was orthotopically transplanted into the liver of 21 nude
mice for studying the ability of secreting AFP from tumor.
Growth
Tumors in the subcutaneous tissue or liver were measured in 3
dimensions with calipers for every 7 days. Tumor size was plotted on
a graph.
Morphology
For light microscopy and transition electron microscopy (TEM) the
primary tumors of liver and xenografts were fixed and stained by the
standard methods.
Assay of AFP, HBsAg and HBcAg
The Sera from patients and tumor bearing mice were analyzed
radioimmunologically with AFP Diagnosis Kit (The Institute of
Biochemical Assay and Product, Shanghai). In brief, 100μL of
blood was obtained weekly for 6 weeks, by orbital venipuncturing
from mice. Two cell lines were inoculated subcutaneously or into
liver (for the later only with hHCC15) respectively. HBsAg and HBcAg
in the sear of tumor-bearing mice were analyzed with HBsAg and HBcAg
Kit (New and Advanced Co. Ltd Xiamen) by ELISA method.
Analysis of chromosome
According to the standard method of preparing metaphase cell treated
with colchicine, the analysis of chromosome in hHCC4 and hHCC15 cell
line were carried out. The number of chromosomes in each spread cell
was counted and all of spread cells were done at least for 100.
Amplification of HBV DNA fragment
The DNA from xenografts were extracted with the standard method of
Sambrook and assayed by the HBV Diagnosis Kit (a kind gift from
professor Wu Bing-Qun, Dept. of Pathology Beijing Medical
University, China). Forward and reverse primer for HBV fragment were
designed as follow: F 5′-GGGTGGAGCCCTCAGGCTCAGGGCA-3,
R 5′-GAAGATGAGGCATAGCAGCAGGAT-3′.
The positive and negative samples were amplified simultaneously as
control. The products of PCR were run in 12g/L agarose gel stained
with ethidimum bromine and photographed.
Amplification of p53 gene fragment
DNA of two strains was a mplified by PCR to produce target of a
110bp at seventh exon of p53 gene using primers of P1 5′-GTTGGCTCTGACTGTACCAC-3′
and P2 5′-CTGGAGTCTTCCAGTGTGAT-3′
on DNA Thermal Cycler 480 (Perkin Elmer/Cetus). The product of 110bp
DNA fragment was identified by 20g/L agarose gel electrophoresis and
DNA dot blot hybridization which was performed with hDIG labeled p53
cDNA probe (2.0kb , cut from reconstructed plasmid ph p53 β,
a kind gift from Professor Liu Si-Li, Tianjin Medical College). The
probe was labeled as the described method of DIG DNA Labeling and
Detection Kit (Boehringer Mannhemi).
Analysis of restriction fragments length polymorphism (RFLP)
Five to 10μl of above PCR amplified products were digested with
7 to 10 unit HaeⅢ
restriction enzyme at 37℃
for 6hr, then precipitated with cooled ethanol. The sediments were
analyzed with 150g/L non-denatural polyacrylamide gel
electrophoresis, and then stained with ethidium bromide and
visualized under UV light.
Sequencing of PCR products
The 110bp of PCR amplification fragments were purified by standard
low-melting point agarose gel electrophoresis method and labeled
with fluorescence according to the description of Tag Dye DeoxyTM
Terminator Cycle Sequencing Kit. The DNA sequence of PCR fragments
were analyzed and edited by Applied Biosystems 373A DNA Sequencer.
RESULTS
Transplantation and growth
The neoplasams of HHC4 and HHC15 were presented in the back of 1 in
6 and all 6 mice respectively and showed rapid growth after several
generations. A 58% and 100% rate of tumor transplantation were
presented in hHCC4 and in hHCC15 respectively. Xenografts were shown
a short latency (18.7 days ±4.9 days and 17.5 days ±1.6 days
respectively) and almost stable after successive generations. The
population doubling time in hHCC4 and hHCC15 cell line was about 7
and 5 days respectively, the growth curves shown in chart 1.
Morphology
Most of the transplanted tumors retained approximately the original
morphological characteristics. Xenografts were shown thick
trabeculae of carcinoma cells possessed increasing nuclear-cytoplasmic
radio (Figure 1). Not any metastasis foci was presented in the
organs of liver and lung in each tumor-bearing mice within 6 weeks.
Ultrastructurally, sinusoid like structure and bile canaliculi were
scattered between two cells (Figure 2). Under TEM, fibrillary
structure of HBsAg could be see in the rough endoplasmic reticulum (RER)
of carcinoma cells in hHCC4 (Figure 3).
Products of AFP, HBsAg and HBcAg
Radioimmunoassay disclosed human AFP in sera of both groups mice
bearing xenografts of both strains, (until as large as 100mm3),
but not for the control group, and their values increased
progressively in relation to orthotopic growth of the tumor in
hHCC15 (Chart 2). HBsAg could be detected in hHCC4, but not for
HBcAg. HBcAg and HBsAg could not be undetected in hHCC15 by ELISA
immunoassay.
Karyology
All evaluable chromosomes of metaphase in hHCC4 and hHCC15 were
human chromosomes, and not any mouse chromosomes were seen (Figure
4). A histogram of chromosome counts of 100 cells disclosed the
number of chromosomes ranging between 50 to 175, the median number
of 106 to 126 in hHCC15 (Chart 3) and the median number of 110 to
134 in hHCC4 (data not shown) cell line respectively.
Figure 1
Light microscope of HHC4 and HHC15 showing high nuclear cytoplasmic
radio. A: HHC4, B: HHC15
Figure
2
The bile canaliculi between two carcinoma cells. ×15000
Figure 3
The filament of HBsAg in the rough Endoplasmic reticulum of hepatoma
cell of HHC4. ×40000
Figure 4
Karyotype of HHC15
Figure 5
Electrophoresis of HBV PCR product from HHC15. A. negative control,
B. HHC15, C. positive control
Figure 6
Dot blot hybridization of p53 PCR product. A. HHC4, B. HHC15, C. ph
p53β plasmid for positive control, D. pBR322 for negative
control
Figure
7
Growth curves of HHC4 and HHC15 in nude mice.
Figure 8 The
relationship between the volume of orthotopical xenograf and the AFP
values in the sera of a nude mouse bearing HHC15.
Figure 9(PDF)
The
histograms of chromosome numbers in HHC15 cell passage 21.
Figure 10
Restriction fragment length polymorphism analysis of PCR products.
A. ph p53β plasmid for positive control, B. DNA marker (pBR322/Hae
Ⅲ),
C. HHC4, D. HHC15
Figure 11(PDF)
Dna
sequencing results of partial PCR fragment showing C→A
mutation at 250 code of p53 gene from HHC4.
Figure 12(PDF)
DNA
sequencing results of partial PCR fragment showing G→T
mutation at 249 code of p53 gene from HHC15.
Amplification of HBV DNA fragment
PCR products amplified from both hHCC4 (data not shown) and hHCC15
or positive control had similar band running in the same distance in
agarose gel, but not any product in negative control sample (Figure
5).
Identification of PCR products and analysis of RFLP
DNA extracted from hHCC4 and hHCC15 xencgrafts and plasmid
ph p53β containing wild p53 cDNA were amplified respectively,
then the PCR products were analyzed by DNA dot blot. Figure 6 showed
positive hybridization of the PCR products from hHCC4, hHCC15 and ph
p53 β
plasmid with the control negative from plasmid pBR322, which meant
the good specificity of the PCR system. With the method of RFLP, it
was shown that the product of PCR from ph p53β plasmid, as a
contrast of wild p53 cDNA, was digested into two bands of 75bp and
35bp (Line A) and undigested two of 110bp from hHCC4 and hHCC15,
displayed respectively on Lane C and Land D (Figure 7). It was
suggested that the mutation at seventh exon of p53 gene could be
presented in the xenografts of hHCC4 and hHCC15.
Sequencing PCR products
The results showed in Figures 8 and 9 were the partial DNA sequence
of fragments amplified from hHCC4 and hHCC15 genomic DNA
respectively. Figure 8 revealed a CCC→ACC
point mutation in 250 code of p53 gene from xenograft of hHCC4,
while Figure 9 showed an AGG→AGT
mutation of p53 gene from xenograft of hHCC15.
DISCUSSION
Xenografts of hHCC in nude mice usually retain their original
morphology, antigen, karyotype and function, such as secreting AFP.
As we know, it has been reported that both AFP and HBsAg can not be
detected simultaneously in nude mouse transplanted with hHCC, but
are presented in the cell lines of hHCC in vitro［3］.
The presenting fibrae of HBsAg in the RER of carcinoma cell support
the specific function of cells from hHCC4.
A
variety of p53 mutations have been found in a wide spectrum of
sporadic tumors, in which the cause of carcinogenesis was still
unknown and direct evidence is absent. It has been reported that
mutation of p53 gene was the most common inducing factor in primary
advanced hHCC. AFB1 was the most inducing factor of
carcinogenesis in hHCC and specially associated with the mutation of
code 249 in p53 gene, being notable as “hot spots” in hHCC,
spreading over South Africa or southeast coast of Asia on the earth［4,5］.
Multiple
line evidence support a closely relationship between AFB1
and HHC. Rats fed with AFB1 developed hepatoma in a
dose-depend fashion［6］.
It has been reported that AFB1 intake might lead the
liver to acute necrosis and proliferation of hepatoid-oval cells［7］.
The more AFB1 intake daily was, the more necrosis
appeared and the oval cells possessing the ability of division
continuously divided.
According
to the telomere hypothesis of cellular senescence theory, somatic
cells, which continuously divided, lead to cell cycle exit and
significant telomere erosion and shortage at which the crisis state
(M1) of cell arrived, which was induced by activating p53
and pRb cascade. It is conceivable recently that p53 are proposed to
signal a growth cheekpoint allowing cell to arrest in G0
or G1 state (replicative senescence) by inducing p21
expression which in turn inactivate cdk/cyclin complex leading to
underphosphorylation of the Rb proteins［8］.
It is reasonable that p53 gene mutation caused by AFB1
have the ability to allow cells to overcome M1, leading
to an extended lifespan until a second growth checkpoint, M2
is reached. This rare event, M2, is most often associated
with the reactivation of telomerase. During the past few years,
there has been mounting evidence that the activation of telomerase,
a ribonucleoprotein enzyme, is important in maintaining telomere
length stability and necessary for the sustained growth of the most
of cancer［9］.
It has been reported recently that due to reactivating telomerase,
mammary epithelial cells, which were transfected with mutation p53
gene, could be survive and become immortal in vitro［10］.
The two cell lines provided molecular biological trace of
relationship between high mortality of hepatoma and AFB1
severe pollution of the daily common food in this district. It was
shown that carcinoma cells of hHCC4 and hHCC15 possessing telomerase
activity (unpublished data) supported the events of p 53 mutation
discovered by us and agree with our suggestion.
The
carcinogenesis of hHCC was shown close relationship of chronic
hepatitis B in which the molecular machanism of hHCC is still a
mystery. Futhermore, AFB1 treatment of trangenic mice
integrated with hepatitis B DNA greatly enhanced the development of
hepatoma as compared with the mice not treated with AFB1［11］.
It is wise to use two cell lines for the disclosing the contribution
of carcinogenesis of human hepatoma in coordination of HBV
integration and p53 mutation which were presented in both of the
cell lines. Also they are the useful material for cell biology,
sensitivity of chemotherapy and gene therapy.
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