Expression of fragile histidine triad in primary hepatocellular carcinoma and its relation with cell proliferation and apoptosis.

Abstract

To evaluate the expression of fragile histidine triad (FHIT) gene protein, product of a candidate tumor suppressor, and to investigate the relationship between FHIT, cell apoptosis and proliferation, and pathological features of primary hepatocellular carcinoma (HCC).
Forty-seven HCC and ten normal liver specimens were collected during surgical operation between 2001 and 2003. FHIT and proliferating cell nuclear antigen (PCNA) expression were detected by immunohistochemistry, and apoptotic level was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay on the tissue sections.
All normal liver tissues showed a strong expression of FHIT, whereas 28 of 47 (59.6%) carcinomas showed a significant loss or absence of FHIT expression (P = 0.001). The proportion of reduced FHIT expression in those carcinomas at stages III-IV (70.6%) and in those with extrahepatic metastasis (86.7%) showed an increasing trend compared with those at stages I-II (30.8%, P = 0.013) and those without metastasis (46.9%, P = 0.010) respectively. Apoptotic incidence in advanced TNM stage carcinoma and those with positive FHIT expression was higher than that in early stage carcinoma (P = 0.030) and in those with negative FHIT expression (P = 0.044) respectively. The proliferating potential of hepatocellular carcinoma was associated with FHIT expression (P = 0.016) and the aggressive feature (P = 0.019). Kaplan-Meier analysis demonstrated that the survival time of these 47 patients correlated with TNM stage, FHIT expression and metastasis.
There is marked loss or absence of FHIT expression, as well as abnormal apoptosis-proliferation balance in HCC. FHIT may play an important role in carcinogenesis and development of HCC.

Full text

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• LIVER CANCER •
Expression of fragile histidine triad in primary hepatocellular
carcinoma and its relation with cell proliferation and apoptosis
Ke-Jun Nan, Zhi-Ping Ruan, Zhao Jing, Hai-Xia Qin, Hong-Yan Wang, Hui Guo, Rui Xu
PO Box 2345, Beijing 100023, China World J Gastroenterol 2005;11(2):228-231
Fax: +86-10-85381893 World Journal of Gastroenterology ISSN 1007-9327
E-mail: wjg@wjgnet.com www.wjgnet.com © 2005 The WJG Press and Elsevier Inc. All rights reserved.
ELSEVIER
Ke-Jun Nan, Zhi-Ping Ruan, Zhao Jing, Hai-Xia Qin, Hui Guo,
Rui Xu, Department of Oncology, First Hospital of Xi’an Jiaotong
University, Xi’an 710061, Shaanxi Province, China
Hong-Yan Wang, Department of Pathology, First Hospital of Xi’an
Jiaotong University, Xi’an 710061, Shaanxi Province, China
Correspondence to: Dr. Ke-Jun Nan, Department of Oncology,
First Hospital of Xi’an Jiaotong University, 1 Jiankang Xilu, Xi’an
710061, Shaanxi Province, China. zoporun@hotmail.com
Telephone: +86-29-85324086 Fax: +86-29-85324086
Received: 2004-02-02 Accepted: 2004-04-07
Abstract
AIM: To evaluate the expression of fragile histidine triad
(FHIT) gene protein, product of a candidate tumor suppressor,
and to investigate the relationship between FHIT, cell apoptosis
and proliferation, and pathological features of primary
hepatocellular carcinoma (HCC).
METHODS: Forty-seven HCC and ten normal liver specimens
were collected during surgical operation between 2001
and 2003. FHIT and proliferating cell nuclear antigen (PCNA)
expression were detected by immunohistochemistry, and
apoptotic level was evaluated by terminal deoxynucleotidyl
transferase-mediated dUTP nick end labeling (TUNEL) assay
on the tissue sections.
RESULTS: All normal liver tissues showed a strong expression
of FHIT, whereas 28 of 47 (59.6%) carcinomas showed a
significant loss or absence of FHIT expression (P = 0.001).
The proportion of reduced FHIT expression in those carcinomas
at stages III-IV (70.6%) and in those with extrahepatic
metastasis (86.7%) showed an increasing trend compared
with those at stages I-II (30.8%, P = 0.013) and those without
metastasis (46.9%, P = 0.010) respectively. Apoptotic
incidence in advanced TNM stage carcinoma and those
with positive FHIT expression was higher than that in early
stage carcinoma (P = 0.030) and in those with negative
FHIT expression (P = 0.044) respectively. The proliferating
potential of hepatocellular carcinoma was associated with
FHIT expression (P = 0.016) and the aggressive feature
(P = 0.019). Kaplan-Meier analysis demonstrated that the
survival time of these 47 patients correlated with TNM stage,
FHIT expression and metastasis.
CONCLUSION: There is marked loss or absence of FHIT
expression, as well as abnormal apoptosis-proliferation balance
in HCC. FHIT may play an important role in carcinogenesis and
development of HCC.
© 2005 The WJG Press and Elsevier Inc. All rights reserved.
Key words: Hepatocellular carcinoma; Fragile histidine triad
protein; Cell proliferation; Apoptosis
Nan KJ, Ruan ZP, Jing Z, Qin HX, Wang HY, Guo H, Xu R.
Expression of fragile histidine triad in primary hepatocellular
carcinoma and its relation with cell proliferation and apoptosis.
World J Gastroenterol 2005; 11(2): 228-231
http://www.wjgnet.com/1007-9327/11/228.asp
INTRODUCTION
Fragile histidine triad gene has been cloned and is located on
3p14.2[1], which encompasses the most common human fragile
site, FRA3B. Alterations of FHIT and loss of its product have
been found frequently in several human tumors and tumor-
derived cell lines associated with environmental carcinogens.
Disturbance of cell number regulation is one of the characteristics
of malignant tumors, which could lead to tumor cell proliferation
out of control. We have known that a variety of oncogenes,
tumor suppressor genes and some modifiers are involved in
the regulation mechanism[2]. Human hepatocellular carcinoma
is a familiar lethal cancer which is closely related to some
carcinogens such as hepatitis B virus infection, dietary aflatoxin
and alcohol consumption. It is imperative to speculate on
whether FHIT, as a putative tumor suppressor gene, plays
a role in the development of hepatocellular carcinoma by
participating in the process of apoptosis or cell cycle. From
this study, the indices of apoptosis and cell proliferation
showed a certain association with the aberrant FHIT expression
in hepatocellular carcinoma, which may elucidate one aspect of
carcinogenesis of malignant tumors.
MATERIALS AND METHODS
M aterials
Forty-seven liver cancer specimens and ten normal liver
specimens as controls were obtained from surgical resections
in the First Hospital of Xi’an Jiaotong University during 2001
to 2003. The patients included 38 men and 9 women with a
mean age of 48.62±10.99 years (range 29-77 years). The
pathological types of all specimens were confirmed tobe
hepatocellular carcinoma by pathologists in Pathology
Department of the First Hospital of Xi’an Jiaotong University.
Of these patients, 39 were at grades I and II, 8 at grade III
according to Edmondson grading and local invasion or
extrahepatic metastasis was observed in 15; and 13 were at
stages I-II, 34 at stages III-IV according to the pTNM criteria of
UICC. The follow-up for all cases was terminated in April of 2004.
Methods
All surgical specimens were fixed in 10% formaldehyde, embedded
in paraffin and cut into 4-µm thick sections. One section of each
specimen was stained with H&E and used for histological
identification, and the rest were used for immunostaining.
Immunohistochemical analysis for FHIT and PCNA
Slides were deparaffinized in xylene twice for 10 min, rehydrated
through graded ethanol to distilled water, incubated for 15 min
with 3% hydrogen peroxidase to inhibit endogenous peroxidase
activity, and then heated in 0.01 mol/L citrate buffer (pH 6.0) in
a microwave oven for 5 min at 100
cooled down at room temperature for 30 min, the sections were
incubated for 15 min in a blocking solution containing 10%
for antigen retrieval. After

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normal goat serum in PBS [0.01 mol/L phosphate (pH 7.4)] and
then incubated for 1 h at 37
rabbit polyclonal antibody to human FHIT (Zymed Laboratories
Inc., South San Francisco, CA) at a dilution of 1:50, followed by
incubation for 30 min with goat anti-rabbit IgG conjugated to
horseradish peroxidase (Santa Cruz Biotechnology). 3,3’-
diaminobenzidine was used as the chromogen. Slides were
counterstained for 3 min with hematoxylin solution, then
dehydrated and coverslipped. Normal liver tissue was used as
a positive control, whereas the primary antibody was replaced
by PBS for a negative control. PCNA was detected by mouse
monoclonal antibody to human PCNA (1:50, Santa Cruz
Biotechnology).
in a humidified chamber with
Apoptosis detection in situ with TUNEL
Paraffin-embedded sections on polylysine-coated slides were
deparaffinized and rehydrated as described in immunohistochemistry
method and then incubated for 15 min with 40 mg/L trypsin at
37
, rinsed in PBS and incubated for 1 h at 37
reaction mixture (buffer solution mixed with terminal
deoxynucleotidyl transferase and nucleotide labeled solution;
Roche, Germany), rinsed again in PBS and incubated for 30
min at 37
with converter-AP. 5-bromo-4-chloro-3-
indalylphosphate/nitro blue tetrazolium (BCIP/NBT) was used
as the chromogen. Slides were counterstained for 5 min with
nuclear fast red solution, then dehydrated and coverslipped.
with TUNEL
Evaluation of scores
Cells filled with yellow or brown granules in cytoplasm were
considered as positive immunostained cells. Both the extent
and intensity of immunostaining were considered when FHIT
protein expression of a section was scored according to Hao
et al.[3]. The extent of positivity was scored as follows: 0, <5%;
1, 5-25%; 2, 25-50%; 3, 50-75%; and 4, >75% of hepatocytes in
respective lesions. The intensity was scored as follows: 0,
negative; 1, weak; 2, moderate; and 3, as strong as in normal
hepatocytes. The final score was obtained by multiplying the
extent of positivity and intensity scores in the range of 0-12.
Scores 8-12 were defined as strong staining, scores 0-6 as markedly
reduced or lost expression.
PCNA positively immunostained cells were filled with brown
granules in nuclei. The apoptotic cells were stained blue in
nuclei, whereas the normal nuclei were stained pink. Regardless
of the extent or intensity of the PCNA and apoptosis staining,
the positive cells were observed and calculated under a light
microscope in 5 high power fields (×400). Proliferation index
(PI) and apoptosis index (AI), expressed as the ratio of positively
stained cells to total cells of the fields, were used to evaluate
the proliferation and apoptosis features respectively.
Statistical analysis
Pearson chi-square test and Fisher’s exact test (two sided) for
trends in proportions were used to assess the association
between FHIT expression and pathological indices. Student’s
t-test was adopted to determine the difference between two
sample means. Survival time was analyzed by Kaplan-Meier
method (SPSS 11.0 for windows). P<0.05 was considered
statistically significant.
RESULTS
FHIT expression in normal tissues and HCC
All the 10 normal liver tissues and para-neoplastic tissues
showed a strong FHIT expression in the cytoplasm of
hepatocytes (Figure 1A). Some lymphocytes and fibroblasts
were positively stained, also. FHIT was expressed in 19
carcinomas as strongly as or more strongly than in normal,
whereas it was expressed negatively in 28 of 47 (59.6%)
carcinomas (Figure 1B). The absence of FHIT expression in
carcinoma was significant (χ2 = 11.709, P = 0.001).
Relationship between FHIT expression and clinicopathological
indices
The proportion of reduced FHIT expression in carcinomas at
stages III-IV was significantly higher (24/34) than that at stages
I-II (4/13) (P = 0.013). This proportion in carcinomas with
extrahepatic metastasis (13/15) was higher than that in those
without metastasis (15/32). No evidence indicated the relation
between FHIT expression and other clinicopathological features
such as age, sex, histological grade and tumor size (Table 1).
Table 1 Relationship between FHIT expression and clinico-
pathological indices of HCC
FHIT score
n P
- +
Age (yr)

>45
Sex
Male
Female
Histological grade
I-II
III
TNM stage
I-II
III-IV
Tumor size (diameter)
<50 mm

50 mm
Extrahepatic metastasis
Positive
Negative
45 18 10
29 18
8
11
0.658
38 21
9 7
18
2
0.278
39 21
8 7
18
1
0.119
13 4
34 24
9
10
0.013
19 12
28 16
7
12
0.680
15 13
32 15
2
17
0.010
AB
Figure 1 FHIT expression in normal tissues and HCC. A: Yellow granules in normal liver tissues, and para-neoplastic tissues; B:
non-stained cytoplasm of tumor cells.
Nan KJ et al. Expression of FHIT in hepatocellular carcinoma229

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Proliferation and apoptosis features in HCC
PCNA was localized in nuclei. Positive PCNA expression manifested
brown granules in nuclei (Figure 2A). Nuclei of apoptotic cells
were stained blue but normal nuclei were stained pink (Figure 2B).
Table 2 summarizes the relationship between AI, PI and
clinicopathological indices. AI showed a similar association with
TNM stage and FHIT expression, i.e., those HCCs at stages III-
IV and those with positive FHIT expression showed a higher AI
compared with those at stages I-II and those with negative FHIT
expression (P<0.05) respectively. PI showed a relationship with
FHIT expression and metastasis (P<0.05).
Survival analysis
The survival time of the forty-seven patients undergoing
surgical resection varied from 1 to 34 mo. Patients with positive
FHIT expression, or those at stages I-II, or those without
metastasis had a longer survival time as shown by Kaplan-
Meier analysis (data not shown).
DISCUSSION
Unregulation of cell proliferation homeostasis results in
unlimited proliferation of malignant cells, which would enlarge
the tumor size and cause cancer. However, its opposite
antagonistic way, apoptosis, determines the cell death. Apoptosis
is an efficacious approach to protect human beings from adverse
carcinogens which may induce mutation or canceration. PCNA
is an auxiliary protein for DNA polymerase delta, which could
perform an essential function in DNA replication and repair
process[4]. The amount of PCNA mRNA varies with DNA
synthesis cycle[5], and reflects the proliferating activity of cancer
cells. Thus, it has been regarded as a proliferation marker for
cancer cells[6,7]. The PCNA labeling index is one useful marker
for evaluating the malignant feature of tumors, and for
predicting recurrence and the prognosis of patients[8].
Apoptosis with necrosis, plays an important role in the cell
life including cell growth, differentiation and proliferation[9]. It is
a regulation mechanism to maintain the homeostasis of tissues.
Malignant tumor tissues lose this regulation and obtain infinite
proliferating potential. There is a controversy concerning the
relationship between proliferation and apoptosis in cancers[10-12].
Our study showed a significant association between PI, AI and
some clinicopathological indices of HCC, such as TNM stage
and extrahepatic metastasis.
FHIT gene spans the most active common fragile site in the
human genome, FRA3B, which is susceptible to inactivation
by environmental carcinogenic factors[13], such as hepatitis B
virus infection, dietary aflatoxin and alcohol consumption[14].
Aberrant FHIT expression has been detected in other tumors,
including lung[15], esophageal[16], stomach[17], colorectal[18],
cervical[19], and prostate[20] carcinomas. Our research
demonstrated a high proportion (59.6%) of reduced FHIT
expression in HCC, compared with positive expression in all 10
normal liver tissues, as reported previously[14]. Moreover, the
aberrant FHIT expression was correlated with TNM stage and
extrahepatic metastasis of HCC. The patients with positive FHIT
expression showed a longer survival time than those with
negative expression. Some findings indicate that loss of FHIT
expression might be an early event in the development of human
carcinoma[16,21]. Therefore, detection of FHIT expression in
malignant tissues by immunohistochemistry may provide some
important diagnostic and prognostic information[22].
FHIT gene consists of 10 exons and encodes FHIT protein
(Mr 16 800), which consists of 147 amino acids. FHIT protein is
a member of histidine triad superfamily and a diadenosine 5’,
5’-P1, P3-triphosphate (Ap3A) asymmetrical hydrolase which
cleaves Ap3A into adenosine 5’-diphosphate and AMP[23].
Siprashvili et al.[24] designed a structure similar to FHIT, in
Figure 2 Expression of PCNA in normal and cancer tissues. A: Positive PCNA expression manifested brown granules in nuclei.
B: nuclei of apoptic cells were stained blue but normal nuclei were stained pink.
AB
Table 2 Relationship between PI, AI and clinicopathological indices (mean±SD)
N AI (%) P value PI (%) P value
FHIT expression
Positive
Negative
Histological grade
I-II
III
TNM stage
I-II
III-IV
Tumor size (diameter)
<50 mm
50 mm
Extrahepatic metastasis
Positive
Negative
19
28
9.42±2.85
8.07±1.63
0.04428.47±4.22
31.61±4.24
0.016
39
8
8.87±2.38
7.38±1.19
0.091 30.46±4.46
29.75±4.74
0.686
13
34
7.46±2.30
9.06±2.15
0.03028.85±3.85
30.91±4.60
0.158
19
28
8.11±2.03
8.96±2.41
0.20829.79±4.85
30.71±4.23
0.492
15
32
9.00±1.89
8.44±2.45
0.43632.53±3.93
29.31±4.38
0.019
230 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol January 14, 2005 Volume 11 Number 2

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which the middle histidine in histidine triad was replaced by
asparagine. This artificial product significantly lost its enzymatic
activity but still maintained tumor suppressor function. It has
been found that hydrolysis for Ap3A is not required for tumor
suppression of FHIT. Researches on the tumor suppression
mechanism of FHIT indicate the active suppressor of FHIT
might function in the form of FHIT-substrate complex[25,26]. Our
study showed a higher AI but a lower PI in carcinomas with positive
FHIT expression, suggesting that FHIT might play a role in the
regulation of cell proliferation and apoptosis. The conceivable
mechanism is that the FHIT-substrate binding complex participates
in signal transduction and induces accumulation of cells at S to
G2-M phase, and then these arrested cells are cleaned out in
manner of apoptosis. This hypothesis is supported by some
molecular experiments[27,28]. Furthermore, some more detailed
researches have revealed that FHIT-induced apoptosis is
practiced in a p53-independent apoptotic pathway[29,30]. This
may provide a new method and opportunity in gene therapy
for malignant tumors.
Carcinogenesis of HCC is a multi-sequential process
involving various oncogenes and tumor suppression genes.
Inactivation of FHIT may play a role in the development and
progression of HCC. FHIT as a valuable target in gene therapy
has a bright future.
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Edited by Wang XL and Zhu LH
Nan KJ et al. Expression of FHIT in hepatocellular carcinoma231