archives of oral biology 53 (2008) 443–452

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Characteristics of a cancerous cell line, HIOEC-B(a)P-96,

induced by benzo(a)pyrene from human immortalized
oral epithelial cell line

Lai-Ping Zhong 1, Hong-Ya Pan 1, Xiao-Jian Zhou, Dong-Xia Ye,

Lei Zhang, Xiao Yang, Wan-Tao Chen, Zhi-Yuan Zhang *
Department of Oral and Maxillofacial Surgery, Ninth People’s Hospital, School of Medicine,
Shanghai Jiao Tong University, Shanghai 200011, China

article info abstract

Article history: Oral squamous cell carcinoma (OSCC) is the most common malignant tumor in the oral and
Accepted 6 December 2007 maxillofacial region. The mechanism of carcinogenesis of OSCC is still unclear. In vitro study
on OSCC cell lines, especially derived from immortalized oral epithelial cells, is a very useful
Keywords: strategy to understand the mechanism of carcinogenesis. Based on our previous human
Oral squamous cell carcinoma immortalized oral epithelial cell (HIOEC) line, obtained from normal oral epithelial cells by
Human papilloma virus type 16 transfection of HPV16 E6/E7 gene, a new cancerous cell line, HIOEC-B(a)P-96 (HB96), was
Benzo(a)pyrene established from the HIOEC by induction with benzo(a)pyrene. The characteristics of the
Carcinogenesis HB96 cells such as cell morphology, ultrastructure, proliferation ability, invasion ability, and
Immortalization tumorigenesis were studied. The HB96 cells lost contact inhibition with uncontrolled cell
division and obvious cell overlap, they were polygonal in shape and ununiform in size with
increased ratio between nucleus and plasma. Increased proliferative ability and invasion
ability were confirmed by the cell proliferation analysis and cell invasion assay, respectively.
The tumorigenicity of well to moderately differentiated squamous cell carcinoma was
confirmed in the nude mice experiments pathologically. Increased expression of HPV16
E6/E7 proteins and obvious correlation with decreased expression of p53 and Rb proteins
was also confirmed by Western blotting. Thus, this HB96 cell line induced by benzo(a)pyrene
from the HIOEC line is a useful tool to study the mechanism of carcinogenesis of OSCC in
vitro for future genomic and proteomic analyses. It is also the first in vitro cancerous cell line
of OSCC in China derived from immortalized oral epithelial cells.
# 2007 Elsevier Ltd. All rights reserved.

1. Introduction Effort has been made to improve the diagnosis and

treatment of OSCC patients, however, the prognosis is still
Oral squamous cell carcinoma (OSCC) is the most common poor with a 5-year survival rate of approximately 50–60%.1,2
malignant tumor in the oral and maxillofacial region. The mechanism of carcinogenesis of OSCC is still not very

* Corresponding author at: Department of Oral and Maxillofacial Surgery, Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong
University, No. 639 Zhizaoju Road, Shanghai 200011, China. Tel.: +86 21 63138341x5385; fax: +86 21 63136856.
E-mail address: zhang.z.y@hotmail.com (Z.-Y. Zhang).
1
These authors contributed equally to this work.
Abbreviations: OSCC, oral squamous cell carcinoma; HIOEC, human immortalized oral epithelial cell; B(a)P, benzo(a)pyrene; HB, HIOEC-
B(a)P; HPV16, human papilloma virus type 16.
0003–9969/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.archoralbio.2007.12.002
444 archives of oral biology 53 (2008) 443–452
clear. In vitro study on OSCC cell lines has been used as an
ideal method to investigate the carcinogenesis mechanism
of OSCC, and many OSCC cell lines have been established
directly from cancerous tissues of OSCC patients. However,
OSCC cell line from normal oral epithelial cells or immor-
talized oral epithelial cells might be better to study the
carcinogenesis mechanism, because it can provide a serial,
gradual, stable, and repeatable progress from normal or
immortalized oral epithelial cell to squamous cell carci-
noma in vitro.
Human papilloma virus (HPV) is a site-specific DNA virus
that is known to infect the basal cell layer and replicate during
epithelial cell differentiation. It has been studied extensively
at the clinical, epidemiological, and experimental levels in the
pathogenesis of OSCC. From a systemic review of HPVs
detection in 5046 head and neck squamous cell carcinomas
worldwide,3 16 types of HPVs have been identified in 2642
OSCCs, the overall HPVs prevalence is 23.5% (95% CI, 21.9–
25.1%). The HPV16 and HPV18 are the most common types in
HPV-positive OSCCs, with the frequency of 68.2% (423/620) on
HPV16 and 34.5% (212/615) on HPV18. Another systemic review
of HPVs detection in OSCCs has reported a higher HPVs
prevalence of 55.5% (76/137), and the most common type is
HPV16.4
Several studies have reported that the integration of HPV16
DNA sequences into normal epithelial cellular DNA can
immortalize the normal epithelial cells into permanent cell
lines.5–8 However, the HPV infection alone does not necessa-
rily lead to malignancy.9
Benzo(a)pyrene [B(a)P], being a kind of polycyclic aromatic
hydrocarbons, is a component of tobacco smoke. Tobacco
smoke is an important cause of OSCC.10,11 The presence of
tobacco-related carcinogen-DNA adducts in oral squamous
cells of smokers, compared with non-smokers, has strength-
ened the link between the development of OSCC and
smoking.12–14 Bioactivation of B(a)P binds to the cellular
DNA in human oral epithelial cells.15 Metabolic activation of
tobacco-associated polycyclic aromatic hydrocarbons like
B(a)P to reactive metabolites and the DNA adducts formed
have been postulated to be central to the carcinogenic process
of polycyclic aromatic hydrocarbon induced cancers.16 How-
ever, the B(a)P alone does not necessarily lead to malignancy,
either; because B(a)P alone fails to transform oral epithelial cell
to malignancy.17
Previous literature has reported an in vitro multistep
carcinogenesis model for OSCC from immortalized normal
human oral keratinocytes to tumorigenic derivative cells,
which may be a useful model for in vitro system investiga-
tion.17 This model has been used for in vitro investigation of
OSCC on differential gene expression, differential mRNA
analysis, JNK signaling pathway, Bmi-1.18–21 Here, based on
our previous human immortalized oral epithelial cell (HIOEC)
line, which had been immortalized from normal epithelial
cells of oral mucosa by transfection of HPV16 E6/E7 gene;8 we
established another cancerous cell line, HIOEC-B(a)P-96,
induced by benzo(a)pyrene from the HIOEC line. It is also
the first in vitro cancerous cell line of OSCC in China derived
from immortalized oral epithelial cells. Furthermore, it would
be a base for us to study the carcinogenesis mechanism of
OSCC in vitro.
2. Materials and methods
2.1. Cell culture and induction by benzo(a)pyrene
The human immortalized oral epithelial cells (HIOECs), which
had been obtained from normal oral mucosa immortalized by
transfection of HPV16 E6/E7 gene as previously described,8
were cultured with defined keratinocyte medium-SFM (Gibco,
USA), and treated intermittently and gradually with 0.1–1.2 mg/
ml benzo(a)pyrene [B(a)P] (Sigma, USA) for 6 months. The cells
were cloned at the 18th passage, and then cultured in DMEM
(Invitrogen, USA) containing 10% fetal bovine serum (FBS)
(Hyclone, USA) from the 21st passage. The cultured cell was
named as HIOEC-B(a)P (HB), the 56th passage of HB was named
as HIOEC-B(a)P-56 (HB56), and the cancerous cell line, the 96th
passage of HB, was named as HIOEC-B(a)P-96 (HB96). The cells
had been cultured in vitro for more than 18 months and more
than 100 passages.
2.2. Assay of cell growth
The HIOECs, HB56 cells, and HB96 cells, were seeded
respectively in a 96-well plate (Corning Costar, Japan) with
1.0  103 cells per well in 200 ml medium (defined keratinocyte
medium-SFM for the HIOEC, and 10% FBS-DMEM for the HB56
and HB96). The cells were subcultured at 37 8C in a 5% CO2
incubator for 1, 2, 3, 4, 5, 6, 7, and 8 days, respectively. At the
end of subculture, 20 ml MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] solution (5 mg/ml in PBS) was
added to each well, and the cells were incubated for further
4 h. Then, the supernatant was removed and 200 ml dimethyl
sulphoxide was added to each well. The plate was shaken at
room temperature for 10 min. Cellular viability was deter-
mined by measuring the absorbance of each well at a
wavelength of 570 nm.
2.3. Cell cycle analysis
The HIOECs, HB56 cells, and HB96 cells, were subcultured for
48 h, and dispersed by trypsinization and suspended in PBS;
then, the cells were stained with propidium iodide using the
Cycle TESTTM PLUS DNA reagent kit (BD Biosciences, USA). The
fluorescence of DNA was measured using a flow cytometer
(FACSCaliburTM, BD Biosciences, USA), and the cell cycle
distribution was analyzed by computer software (ModFit LTTM,
BD Biosciences, USA). The procedure was performed according
to the manufacturer’s protocol.
2.4. Immunocytochemistry
The procedure was performed as previously described.8 Briefly,
the HB96 cells were subcultured on sterilized glass coverslips,
when the cells were 70–80% confluent, they were fixed in 4%
paraformaldehyde in PBS (pH 7.4), permeabilized with 0.1%
Triton X-100 in PBS, and subjected to immunostaining using
primary anti-cytokeratin pan AE1/AE3 antibody (dilution: 1/50)
(Dako Cytomation, Denmark) and primary anti-vimentin anti-
body (dilution: 1/50) (Sigma, USA), respectively. After incubation
with primary antibody, biotinylated second antibody was added
followed by streptavidin conjugated horseradish peroxidase.
 archives of oral biology 53 (2008) 443–452
Immunoreaction was visualized using 3,30 -diaminobenzidine
detection kit (Dako Cytomation, Denmark).
2.5. In vitro invasion ability
The HIOECs, HB23 cells, HB56 cells, and HB96 cells, were plated
at a density of 2.0  105 cells per well in a 24-well culture plate,
four wells for each HB passage. The invasion assay was done
using a transwell cell culture system (Corning Costar, Japan).
The upper surface of transwell insert with 8 mm pore size was
coated with 100 mg/ml Matrigel (BD Biosciences, USA). The
cells at a density of 2  105 were seeded in the upper chamber
of each well with 100 ml serum-free DMEM. 600 ml 10% FBS-
DMEM was placed in the lower chambers. The cells were
allowed to migrate at 37 8C in a 5% CO2 incubator for 20 h.
Then, the cells were fixed in methanol and stained with
Giemsa. Migration was determined by the count of the cells
that had migrated to the lower surface of the insert with a
microscope at 400 magnification. Five random visual fields
were counted for each well, and the average was determined.
2.6. Western blot analysis
The procedure was also performed as previously used.8 Briefly,
proteins (50 mg/lane) were separated by 12% SDS-PAGE for 2 h.
After electrophoresis, proteins were transferred onto polyviny-
lidenedifluoride (PVDF) membrane (Hybond-P, Amersham,
USA) for 3 h at 120 mA. The membrane was blocked with 7.5%
skimmed milk in the solution of PBS with 0.1% Tween 20 (PBST)
for 1 h. After being washed three times with PBST, the
membrane was incubated with primary antibody diluted in
PBST at room temperature with agitation for 3 h. After being
washed three times with PBST, the membrane was incubated
with secondary horseradish peroxidase-conjugated antibody
(Santa Cruz Biotechnology, USA) at room temperature with
agitation for 2 h, and then washed three times with PBST.
Protein bands were detected by chemiluminescence with ECL
PlusTM Western Blotting Detection Reagents (GE Healthcare,
USA). b-actin was used as internal control protein. The following
antibodies were used: anti-E6 antibody (dilution 1:200) (C1P5,
Santa Cruz Biotechnology, USA), anti-E7 antibody (dilution
1:200) (TVG-701Y, Oncogene Science, USA), anti-p53 antibody
(dilution 1:300) (PAb 240, Oncogene Science, USA), anti-Rb
antibody (dilution 1:300) (Ab-6, Oncogene Science, USA), and
anti-b-actin antibody (dilution 1:10,000) (AC-15, Sigma, USA).
2.7. Tumorigenicity
The cells, including the HB19, HB28, HB38, HB42, HB50, HB56,
HB69, HB74, and HB96, were trypsinized, resuspended in PBS
at a concentration of 4.0  107 cells/ml, and then 0.2 ml of this
suspension was injected into two sites of SPF BALB/c nude
mice (nu/nu, aged 4–5 weeks, and weighted 18–22 g) sub-
cutaneously. Six mice were used for each HB passage
mentioned above. Mice were monitored for tumor develop-
ment every week after the injection for 3 months. After 3
months, the mice were killed. The tumors were measured in
size, and then fixed in 10% neutralised formalin, and
embedded in paraffin. Each sample was studied using the
hematoxylin and eosin (HE) staining. All animal studies were
445
approved by the Animal Research Committee of the Ninth
People’s Hospital, School of Medicine, Shanghai Jiao Tong
University.
2.8. Statistical analysis
All data was analyzed using the statistical software package
SPSS for Windows version 10.0 (SPSS Inc., USA). The results
were expressed as means  S.D. for at least triplicate
determinations. The initial data was first tested for the
distribution using one-sample Kolmogorov–Smirnov test. If
the distribution was normal, parametric tests were performed;
if the distribution was not normal, nonparametric tests were
performed. When the P-value was less than 0.05, the
difference was regarded as statistically significant.
3. Results
3.1. Cell morphology
After the HIOECs were treated with B(a)P for 6 months, the HB
cells had been cultured in vitro for more than 18 months and
over 100 passages in 10% FBS-DMEM. The HIOECs and HB96
cells were subcultured on sterilized glass coverslips, and then
they were fixed in 4% paraformaldehyde in PBS (pH 7.4). Using
the differential interference contrast microscope (DICM), the
morphology of the HIOECs was cobble stone-like in shape, the
cells showed parallel arrangement with no tendency toward
overlapping, and the cells were almost uniform in size
(Fig. 1A). Using the HE staining, the HIOECs showed the
character of polygonal epithelial cells, with proper ratio
between nucleus and plasma, and proper cell division
(Fig. 1B). For the HB96 cells, with the loss of contact inhibition,
the cells showed uncontrolled cell division with overlapping
using the DICM (Fig. 1C). Using the HE staining, the HB96 cells
also showed the character of epithelial cells, however, the
increased ratio of nucleus and plasma, more cell division, and
sporadic tumor giant cells showed the malignant character
(Fig. 1D).
3.2. Cell ultrastructure
Using the transmission electron microscope (TEM), the
HIOECs showed rich, thick, and compact tonofilaments in
their cytoplasm (Fig. 1E and F). Using the scanning electron
microscope (SEM), the HIOECs showed the character of
polygonal epithelial cells, with proper ratio between nucleus
and plasma (Fig. 1G). For the HB96 cells, the nucleus was
enlarged and irregular with more euchromatin and multiple
nucleoli, the tonofilaments were short, thin, and sparse
(Fig. 1H and I) using the TEM. Using the SEM, the HB96 cells
were polygonal and ununiform in size with more cytoplasmic
processes and microvilli on the cells’ surface (Fig. 1J).
3.3. Analysis of cell growth
From the cell growth curve by MTT assay (Fig. 2A), the HB96
cells grew obviously faster than HB56 cell and HIOECs, and the
HB56 cells grew faster than the HIOECs.
446 archives of oral biology 53 (2008) 443–452

Fig. 1 – The morphology of HIOECs and HB cells. (A) The HIOECs were cobble stone-like in shape (DICM, magnification 200T);
(B) the HIOECs were polygonal epithelial cells (HE staining, magnification 400T); (C) the HB cells were overlapped with loss
of contact inhibition (DICM, magnification 200T); (D) the HB cells were epithelial cells with increased ratio of nucleus and
plasma, more cell division, and sporadic tumor giant cells (HE staining, magnification 400T); (E–J) the ultrastructures of
HIOECs and HB cells using TEM and SEM; (E–G) the HIOECs were polygonal, the tonofilaments were rich, thick, and compact
(white arrow); (H–J) the HB cells were also polygonal and ununiform in size with more cytoplasmic processes and microvilli
on the cells’ surface; however, the tonofilaments were short, thin, and sparse (black arrow) (TEM magnification 4000T).

3.4. Cell cycle analysis
In order to confirm the MTT results, the cell cycle of the
HIOECs, HB56 cells, and HB96 cells was analyzed using a flow
cytometer (Fig. 2B). A tendency of increased proliferation of
cells from the HIOECs to HB56 cells, and then to HB96 cells was
found with increase of percentage of cells in the S phase and
G2/M phase and decrease of percentage of cells in the G0/G1
phase. In the HB96 cells, the percentage of cells in the S phase
(40.95%) and G2/M phase (23.59%), also known as proliferative
index, was higher than that in the HB56 cells (S phase, 32.25%;
G2/M phase, 22.75%) and HIOECs (S phase, 14.07%; G2/M phase,
19.09%). However, in the HB96 cells, the percentage of cells in
the G0/G1 phase (35.47%) was lower than that in the HB56 cells
(44.99%) and HIOECs (66.84%).
3.5. Immunocytochemistry
Using the immunocytochemistry, strong positive cytokeratin
pan AE1/AE3 expression and week positive vimentin expression
 archives of oral biology 53 (2008) 443–452 447

Fig. 2 – (A) The cell growth curves of HIOECs, HB56 cells, and HB96 cells. The HB96 cells grew fastest, the HIOECs grew
slowest; (B) cell cycle distribution of the HIOECs, HB56 cells, and HB96 cells using a flow cytometer. An increased tendency
of cell proliferation from the HIOECs to HB56 cells, and to HB96 cells were found with increase of percentage of cells in the S
phase and G2/M phase and decrease of percentage of cells in the G0/G1 phase.

of the HB96 cells indicated that the HB96 cells were epithelial cells, to HB56 cells, and then to HB96 cells was found (ANOVA,
differentiated (Fig. 3). F = 66.779, d.f. = 3, P < 0.001) (Fig. 4).

3.6. In vivo invasion ability 3.7. Tumorigenicity

All cells grew well in the transwell chamber. After the cells
were stained with Giemsa, the count of cells that had migrated
to the lower surface of the insert was determined. The
migrated cells were all epithelioid cells (Fig. 4). No migration of
the HIOECs was found. The count of the migrated HB23 cells
was 1.8  1.3 per visual field; the count of the migrated HB56
cells was 6.8  0.8 per visual field; the count of the migrated
HB96 cells was 11.4  2.3 per visual field. An increased
tendency of migrated cell amount from the HIOECs to HB23
In order to assess the tumorigenicity in the nude mice, the HB
cells (including HB19, HB28, HB38, HB42, HB50, HB56, HB69,
HB74, and HB96) were injected subcutaneously at two sites of
the mice. After the mice were killed, no tumor was found in
the mice injected with the HB19, HB28, HB38, HB42, and HB50
cells. In the mice injected with the HB56 cells, hard neoplasm
was found subcutaneously about 0.3  0.1 cm in size; the rate
of neoplasm formation was 16.7% (1/6). Using the HE staining,
the neoplasm had intact envelope with 2–3 layers of epithelial
448 archives of oral biology 53 (2008) 443–452

Fig. 3 – The HB96 cells were strong positive staining for cytokeratin pan AE1/AE3 and weak positive staining for vimentin.
(A) Anti-cytokeratin pan AE1/AE3 staining (magnification 200T); (B) anti-cytokeratin pan AE1/AE3 staining (magnification
400T); (C) anti-vimentin staining (magnification 200T); (D) anti-vimentin staining (magnification 400T).

cells under the envelope; under the epithelial cells, massive
keratinocytes with keratinization were found, parakeratosis
was also found in some keratinocytes with few atypical
hyperplasia (Fig. 5A and B). In the mice injected with the HB69
cells, the rate of neoplasm formation was 50.0% (3/6), the
neoplasm was about 0.5  0.1 cm in size. Using the HE
staining, the neoplasm was squamous cell carcinoma patho-
logically; the edge of the tumor was clear, which was
composed of several layers of epithelial cells. The differentia-
tion of the tumor cells was good with some atypical
hyperplasia, pathological mitoschisis, and parakeratosis
(Fig. 5C and D). In the mice injected with the HB74 cells, the
rate of neoplasm formation was 66.7% (4/6), the neoplasm was
about 0.6  0.2 cm in size. Using the HE staining, the neoplasm
was typical squamous cell carcinoma; the differentiation of
the tumor cells was good (Fig. 5E and F). In the mice injected
with the HB96 cells, the rate of neoplasm formation was 83.3%
(5/6), the neoplasm was about 1.0  0.2 cm in size. Using the
HE staining, the neoplasm was also typical squamous cell
carcinoma; the differentiation of the tumor cells was good to
moderate with obvious atypical hyperplasia and pathological
mitoschisis (Fig. 5G and H).
3.8. Western blot analysis of HPV16 E6/E7, p53, and Rb
proteins
In order to confirm the continuous expression of the HPV16 E6
and E7 proteins in the HIOECs, HB56 cells, and HB96 cells, and
to investigate the expression of p53 and Rb proteins in these
cells, Western blotting was performed. As we previously
described, the HIOEC was established from normal oral
epithelial cells by transfection with the HPV16 E6 and E7
gene.8 Here, the normal epithelial cells, which were used as
control, were obtained from the patients undergoing surgery
for cleft plate or lip reconstruction as previously described.8
The relative quantification (logarithm) of the HPV16 E6 and E7
proteins to the internal control protein, b-actin, in the HIOECs,
HB56 cells, and HB96 cells was determined compared with
those in the normal oral epithelial cells. The increased
expression of HPV16 E6 and E7 proteins was all found in the
HIOECs, HB56 cells, and HB96 cells. Furthermore, the expres-
sion level of HPV16 E6 protein was obviously higher than that
of HPV16 E7 protein (Fig. 6). The decreased expression of p53
and Rb proteins was found in the HIOECs, HB56 cells, and HB96
cells by different degree. The decreased expression of Rb
protein in the HB96 cells was more obvious than that of p53
and Rb proteins in the other cells (Fig. 6).
4. Discussion
Our results show the characteristics of a cancerous cell line,
HIOEC-B(a)P-96 (HB96), induced by B(a)P from the HIOEC,
which has been established from normal oral epithelial cells
by transfection with HPV16 E6/E7 gene.8 The HB96 cells lose
the contact inhibition with uncontrolled cell division and
obvious cell overlap, which is different from the HIOECs.
The shape of the HB96 cells is polygonal and the size is
ununiform. The nucleus is enlarged and irregular with more
euchromatin and multiple nucleoli. Increased ratio between
nucleus and plasma, more cell division, and sporadic
tumor giant cells can also be observed. These results
support the malignant character of HB96 cells. Using the
immunocytochemistry, the HB96 cells are strong positive for
 archives of oral biology 53 (2008) 443–452 449

Fig. 4 – After the cells were stained with Giemsa, the count of migrated cells could be determined. The migrated cells were all
epithelioid cells. No migration of HIOECs was found. The count of migrated HB23, HB56, and HB96 cells was 1.8 W 1.3,
6.8 W 0.8, and 11.4 W 2.3 per visual field, respectively. An increased tendency of migrated cell amount from the HIOECs to
HB23 cells, to HB56 cells, and to HB96 cells was found (ANOVA, F = 66.779, d.f. = 3, P < 0.001).

cytokeratin pan AE1/AE3 and weak positive for vimentin,
which indicates the epithelial differentiation, not obvious
mesenchymal differentiation.22,23
The HB96 cells grow faster than the HB56 cells and HIOECs
in the same condition detected by MTT assay. The increased
tendency of the proliferative index (the percentage of cells in
the S phase and G2/M phase) and the decreased tendency of
the percentage of cells in the G0/G1 phase from the HIOECs, to
HB56 cells, then to HB96 cells also indicates the increased
proliferative ability of the HB96 cells compared with the HB56
cells and HIOECs.
Using the in vitro invasion experiment, an increased
tendency of cell invasion ability can be found from the HIOECs
to HB56, and then to HB96 cells. Furthermore, the tumor-
igenicity experiment confirms the tumorigenicity of the HB96
cells in the nude mice with the neoplasm formation rate of
83.3% (5/6). Using the HE staining, the tumors are confirmed as
well to moderately differentiated squamous cell carcinoma,
which is almost same to the clinical oral pathological
examination.
The continuous and stable expression of the HPV16 E6 and
E7 proteins in the HIOECs, HB56 cells, and HB96 cells confirms
the successful integration of the HPV16 E6 and E7 gene into the
normal oral epithelial cells as previous described.8 Park et al.17
have reported that the sequential exposure of oral keratino-
cytes to HPV16 DNA and B(a)P can convert normal cells to
tumorigenic cells in vitro. In the present study, under the
cooperation of HPV16 E6/E7 gene and B(a)P, the HIOECs are
also converted to the tumorigenic cells, the HB96 cells. The
tumorigenicity of the HB96 cells is also confirmed by the
subcutaneous squamous cell carcinoma in the nude mice. As
known to all, a causal role of HPV16 in carcinogenesis is linked
to the activity of the viral oncoproteins E6 and E7 which
inactivate the cellular tumor suppressors of p53 and Rb,
respectively.24–30 In this study, the increased expression of the
HPV16 E6 and E7 proteins in the HIOECs, HB56 cells, and HB96
450 archives of oral biology 53 (2008) 443–452

Fig. 5 – Subcutaneous neoplasms in the mice injected with the HB56, HB69, HB74, and HB96 cells. (A and B) Injection with the
HB56 cells; (C and D) injection with the HB69 cells, well-differentiated squamous cell carcinoma; (E and F) injection with the
HB74 cells, well differentiated squamous cell carcinoma; (G and H) injection with the HB96 cells, well-moderately
differentiated squamous cell carcinoma (A, C, E, G: HE staining, magnification 40T; B, D, F, H: HE staining, magnification
100T).

cells also correlates with the decreased expression of the p53
and Rb proteins, respectively. Alternation of p53 expression is
common in carcinogenesis. The decreased expression of p53
has been reported to be involved in the development of oral
malignancy, not only in OSCC cell lines, but also in the multi-
step oral carcinogenesis model,17,31,32 it has also been reported
to be involved in the expression alternation of apoptosis-
related molecules, such as Bax, Bcl-2, Fas-R, and FAP-1.32
This cancerous cell line of HB96 is a useful cell line to
investigate the formation mechanism of OSCC in vitro. Using
the HIOECs, HB56 cells, and HB96 cells as an in vitro
carcinogenesis model of OSCC, further comparison on the
differential gene and protein expressions could be per-
formed. It could be possible to find out a group of genes and
proteins, which contributes the carcinogenesis of OSCC in
vitro.
 archives of oral biology 53 (2008) 443–452 451

Fig. 6 – Using Western blot analysis, increased expression of the HPV16 E6/E7 proteins and decreased expression of the p53
and Rb proteins were found in the HIOECs, HB56 cells, and HB96 cells. N = normal oral epithelial cells; Actin = b-actin.
Relative quantification (logarithm) of the HPV16 E6/E7, p53, and Rb proteins to internal control protein, b-actin, in the
HIOECs, HB56 cells, and HB96 cells was determined compared with those in the normal oral epithelial cells. The increased
expression of the HPV16 E6 protein in the HIOECs, HB56 cells, and HB96 cells was obviously higher than that of the HPV16
E7 protein. The decreased expression of Rb protein in the HB96 cells was more obvious than that of p53 and Rb proteins in
the other cells. The increased expression of the HPV16 E6 and E7 proteins correlates with the decreased expression of the
p53 and Rb proteins, respectively.

Acknowledgements after transfection of human keratinocytes with human

This work was supported by research grants from National
Natural Science Foundation of China (30630065 and 30700953),
Science and Technology Commission of Shanghai Munici-
pality (05dz22319), China Postdoctoral Science Foundation
(20060400168), and Shanghai Educational Development Foun-
dation (2007CG22).
Conflict of interest
None.
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