Ki-67 Expression in Gingival Overgrowth: An Immunohistochemical Study

Abstract

Ki-67 is a well-recognized nuclear proliferation marker. Considering that an unusual cell proliferation may have a role in the pathogenesis of gingival overgrowth with different etiologies. The study involved 4 patients with cyclosporine induced gingival overgrowth (CGO), 6 patients with phenytoin induced GO (PGO) and 5 patients with hereditary gingival fibromatosis (HGF). Healthy tissue samples without clinical signs of periodontal inflammation were also included as control samples. Immunohistochemistry against the proliferation antigen Ki-67 was performed and optical density measured and compared in both epithelium and connective tissue. Ki-67 was expressed both in the epithelium and corium of the four studied groups. The expression patterns of Ki-67 were significantly higher (p<0.00) in CGO, while no significant difference between HGF and PGO groups was detected and both showed lower values than CGO. Control group showed the significantly lowest mean of Ki-67 level and the expression was mainly in the basal layer of epithelium. In conclusion; increased cell division may have a role in the pathogenesis of gingival overgrowth induced by cyclosporine and phenytoin or inherited as HGF as reflected by increased expression of Ki-67.

Full text

Life Science Journal, 2011;8(X) http://www.lifesciencesite.com
http://www.sciencepub.net/life lifesciencej@gmail.com
221
Ki-67 Expression in Gingival Overgrowth: An Immunohistochemical Study
Eman Y. El-Firt *1 and Dalia M. Ghalwash 2
1Departement of Oral Medicine and Periodontology, Faculty of Oral and Dental Medicine, Cairo University,
Cairo, Egypt
2Departement of Oral Medicine and Periodontology, Faculty of Dentistry, October University for Modern
Sciences and Arts, 6th October, Egypt
*elfirt@yahoo.com

Abstract: Ki-67 is a well-recognized nuclear proliferation marker. Considering that an unusual cell proliferation
may have a role in the pathogenesis of gingival overgrowth with different etiologies. The study involved 4
patients with cyclosporine induced gingival overgrowth (CGO), 6 patients with phenytoin induced GO (PGO)
and 5 patients with hereditary gingival fibromatosis (HGF). Healthy tissue samples without clinical signs of
periodontal inflammation were also included as control samples. Immunohistochemistry against the proliferation
antigen Ki-67 was performed and optical density measured and compared in both epithelium and connective
tissue. Ki-67 was expressed both in the epithelium and corium of the four studied groups. The expression
patterns of Ki-67 were significantly higher (p<0.00) in CGO, while no significant difference between HGF and
PGO groups was detected and both showed lower values than CGO. Control group showed the significantly
lowest mean of Ki-67 level and the expression was mainly in the basal layer of epithelium. In conclusion;
increased cell division may have a role in the pathogenesis of gingival overgrowth induced by cyclosporine and
phenytoin or inherited as HGF as reflected by increased expression of Ki-67.
[Eman Y. El-Firt and Dalia M. Ghalwash Ki-67 Expression in Gingival Overgrowth: An
Immunohistochemical Study] Life Science Journal,. 2011; 8(4):221-226] (ISSN: 1097-8135).
http://www.lifesciencesite.com.
Keywords: Cyclosporine; phenytoin; hereditary gingival fibromatosis; gingival hyperplasia/pathogenesis; Ki-67
1. Introduction
Gingival enlargement may be caused by a
variety of etiologic factors. Some drugs such as
cyclosporin A; the drug of choice in preventing
transplant rejection; and phenytoin; the most
commonly used drug for managing epileptic
seizures; are commonly associated with the
adverse effect of gingival overgrowth (Rateitschak
et al., 1983; Bulut et al., 2004; Lin et al., 2007).
Gingival overgrowth can also be inherited as an
autosomal dominant disorder, or occasionally as an
autosomal recessive mode of inheritance, in a
condition known as hereditary gingival
fibromatosis (HGF) (Singer et al., 1993; Coletta
and Graner, 2006). Both types of gingival
overgrowth (drug induced or familial) are
characterized histologically by thickened,
parakeratinized epithelium with elongated rete-pegs
and increased extracellular matrix within the
connective tissue (Mariani et al., 1993; Martelli et
al., 2000; Vardar et al., 2005).
Squamous cell carcinomas may arise in some
cases of drug induced gingival hyperplasia although
it has been long thought that these conditions are
not related to tumorigenesis (Varga and Tyldesley,
1991; McLoughlin et al., 1995; Saito et al., 1999).
Oral cancers and increased proliferative activity of
oral tissues have been analyzed for many years by
monoclonal antibodies to specific antigens such as
Ki-67 (Zoeller et al., 1994). Ki-67 is a proliferation
associated antigen that serves as a marker for
estimation of tissue growth as it is present in the
nuclei of proliferating cells located in G1, S, G2,
and M phases of the cell cycle and absent in
quiescent cells lagging in G0 phase, suggesting a
role for Ki-67 in the early steps of rRNA synthesis
(Schlter et al., 1993; Buduneli et al., 2007). The
mean rate of ki-67 positive cells in phenytoin-
induced gingival overgrowth is proven to be more
than 10% of immune-stained sections, which is
comparable to that of dysplastic oral mucosae
(Saito et al., 1999).
As for the HGF, although they usually
represent a totally benign condition, yet one case of
epithelial dysplasia of the overgrown tissue has
been reported (Gunhan et al., 1995). HGF epithelial
cells demonstrated higher proliferation rates than
normal gingivae and increased expression of
proliferation markers as proliferating cellular
nuclear antigen (PCNA) and Ki-67 of HGF
mesenchymal fibroblasts has been detected in vitro
(Saygun et al., 2003; Martelli et al., 2005).
The present study aimed to evaluate the state
of imbalance in homeostasis of the proliferative
activity of gingival epithelium and connective
tissue cells by comparing the immunohistochemical
expression of a commonly used proliferation
marker, Ki-67, in cyclosporine and phenytoin
gingival overgrowth as well as cases of HGF.

Life Science Journal, 2011;8(X) http://www.lifesciencesite.com
http://www.sciencepub.net/life lifesciencej@gmail.com
222
2. Material and Methods
Study Population
Gingival biopsies were collected from 15
subjects (seven females and eight males with age
ranges from 10-32 yrs) with moderate to severe
gingival overgrowth (GO) during gingivectomy
procedures. The study involved 4 patients with
cyclosporine induced gingival overgrowth (CGO), 6
patients with phenytoin induced GO (PGO) and 5
patients with hereditary gingival fibromatosis
(HGF). Diagnosis was based on patients’ history and
clinical examination to differentiate between
different causes of gingival overgrowth shown in
figure 1 (A, B and C).
Healthy tissue samples without clinical signs of
periodontal inflammation were also obtained from
the marginal gingiva of four unrelated HGF patients
(two males and two females, aged 16 - 28 yrs) when
the subjects underwent routine dental treatment
(tooth extraction for orthodontic reasons or crown-
lengthening procedures). All patients signed a
consent form after being advised of the nature of the
study.
Figure (1): Clinical view of three patients with
severe gingival overgrowth: A: CGO, B: PGO and
C: HGF
Tissue Processing
As previously described by Buduneli et al.,
(2007), tissue samples were fixed in 4%
paraformaldehyde and embedded in paraffin.
Sections with 5-µm thickness were cut at the central
region of each specimen to obtain maximum
standardization of the cutting surface. One of the
sections was stained with hematoxylin and eosin to
evaluate the histopathologic presentation of gingival
enlargement.
For Ki-67 staining, sections were
deparaffinized by passing through xylene and
alcohol, and rehydrated in 96% ethanol, then
immersed in 3% hydrogen peroxide to block
endogenous peroxide activity. The sections were
incubated with a mouse anti-human Ki-67 antibody
(Zymed, CA, USA) at 4°C overnight. Normal serum
was used as a negative control. Subsequently, the
standard streptavidin–biotin–peroxidase complex
method was performed using SP kit (Zhongshan
Goldenbridge Biotechnology, Beijing, China) for
immunohistochemical detection of the proliferation
marker Ki-67. Reaction products were visualized by
immersing the sections for 5 min in
diaminobenzidine solution. Nuclei were lightly
counterstained with hematoxylin. Each step was
followed by thorough washes with phosphate
buffered saline (PBS).
Assessment of immunostaining
Ordinary light microscope was first used to detect
the positive and negative immunostaining and
localization of the positive reaction within the
tissues. Image analyzer computer system (Leica
Qwin 500 image analyzer computer system,
Wetzlar, Germany) was used to measure the optical
density (OD) of the immunostained sections. Five
sections were used for each subject and three fields
of a gingival section were chosen randomly for the
analysis of Ki-67 staining using a magnification of
(x400) so that a total of 15 microscopic fields were
analyzed for each subject.
Statistical Analysis
Data were presented as mean and standard deviation
(SD) values. One-way Analysis of Variance
(ANOVA) was used for comparison between the
four groups. Tukey’s post-hoc test was used for pair-
wise comparison between the groups when ANOVA
test is significant. Paired t-test was used to compare
between Ki-67 levels in epithelium and connective
tissue. The significance level was set at P ≤ 0.05.
Statistical analysis was performed with PASW
Statistics 18.0 (Predictive Analytics Software) for
Windows (IBM Company, Chicago, IL, USA).
3. Results

Life Science Journal, 2011;8(X) http://www.lifesciencesite.com
http://www.sciencepub.net/life lifesciencej@gmail.com
223
Histopathology and Immunohistochemistry
As shown in H&E stained sections in figure 2
(A&B), the histopathological features did not differ
greatly between different cases of drug induced GO
(CGO and PGO). They shared a common
histopathology of a significant papillary hyperplasia
and parakeratinized epithelial layer with acanthosis
and deep ridges penetrating into the underlying
connective tissue, with wide variable levels of
inflammatory cell infiltration and chorion fibrosis.
Connective tissue alterations were more
marked in specimens from HGF group manifested
by increased amount of collagen fiber bundles and
fewer fibroblasts. Mild chronic inflammatory
infiltrates were also frequently observed in the
subepithelial connective tissue samples. These
changes are shown in fig (3).
Nuclear immunoreactivity for Ki-67 antigen
was easily identified, and nuclei with a clear brown
color, regardless of the intensity of staining, were
interpreted as positive, but this positive reaction was
more marked and widely distributed within
epithelial cells than within connective tissue cells. In
healthy control tissues, Ki-67-positive cells were
observed only in the basal cell layer of epithelium
while the majority of gingiva samples from the CGO
group showed deep and widely distributed Ki-67
positive cells throughout epithelial layers. The PGO
and HGF groups showed almost similar expression
of Ki-67 antigen which was mainly located in the
basal and suprabasal layers of the epithelium. In the
lamina propria, Ki-67 expression was observed in
fibroblasts of hyperplastic gingival tissues mainly in
tissue sections belonging to the CGO, while the
control gingiva revealed week immunostaining of
fibroblasts. These findings of the immune stained
sections are shown in figure (4).
Figure (2): Histopathologic presentations of drug induced GO: A - Section from CGO showing irregular
acanthosis and chorion fibrosis with marked inflammatory cellular infiltrate. B - Section from PGO
group showing acanthosis with mild parakeratosis and papillomatosis with chorion fibrosis and
lymphomononuclear infiltrate (H&E; original magnificationx100).
Figure (3): Histopathologic presentation of HGF showing dense connective tissue predominantly
consisting of thick and irregularly arranged collagen fibers underlying a well structured
epithelium (H&E; original magnification x100).

Life Science Journal, 2011;8(X) http://www.lifesciencesite.com
http://www.sciencepub.net/life lifesciencej@gmail.com
224
Figure (4): Ki-67 antigen expression in tissue samples of all study groups.
A: Ki-67 antigen –positive nuclei observed mainly in the basal layer of control gingival epithelium. B:
Deep immune staining of the nuclei of almost all layers of the hyperplastic epithelium in CGO. C:
immune stained section of PGO and D: HGF showing comparable patterns of Ki-67 antigen
expression in the basal and suprabasal layers of the epithelium. Note that in all sections connective
tissue expression of Ki-67 antigen is less marked than epithelium (immunostaining; original
magnification x200).
Optical Density
As shown in table (1), Ki-67 was expressed
both in the epithelium as well as the corium of the
four studied groups with the epithelium showing
the significantly higher means of Ki-67 OD than
connective tissue in CGO and PGO groups as well
as control tissue samples at P values of 0.003, 0.006
and 0.014; respectively. As for HGF group, there
was no significant difference in the mean values of
Ki-67 OD between epithelium and connective
tissue (P=0.747).
As shown in table (2), The OD of Ki-67 in the
keratinocytes within CGO group showed the
significantly highest mean of Ki-67 level (73.4±4.4)
at a P value <0.001. There was no significant
difference between HGF and PGO groups; both
showed lower values than CGO, while control
tissue samples showed the lowest mean of Ki-67
level (33.4±11.8). Similar findings were detected in
the corium of the test and control groups with the
significantly highest mean of Ki-67 seen in the
corium of CGO group (60.4±0.4).
Table (1): The mean, standard deviation (SD) values and results of paired t-test for comparison between
Ki-67 OD levels in epithelium and connective tissue within each group
Control HGF PGO CGO
Epithelium 33.4±11.8 46.7± 2.9 50.9±2.1 73.4±4.4
Connective tissue 16±7.9 45±10.6 30.9± 9 60.4±0.4
P-value 0.014* 0.747 0.006* 0.003*
*: Significant at P ≤ 0.05

Life Science Journal, 2011;8(X) http://www.lifesciencesite.com
http://www.sciencepub.net/life lifesciencej@gmail.com
225
Table (2): The mean, standard deviation (SD) of optical density values and results of ANOVA test for
comparison between Ki-67 levels in the four groups
Control HGF PGO CGO P-value
Epithelium 33.4±11.8c 46.7±2.9b 50.9±2.1b 73.4±4.4a <0.001*
Connective tissue 16±7.9c 45±10.6
b
30.9±9
b
60.4±0.4a <0.001*
*: Significant at P ≤ 0.05, Means with different letters are statistically significantly different according to Tukey's test
4. Discussion
Although it has been thought that drug-
induced gingival hyperplasia is not related to
tumorigenesis, recent case reports have shown that
squamous cell carcinoma may arise in gingival
hyperplasia induced by cyclosporine (Varga and
Tyldesley, 1991) and phenytoin (McLoughlin et al.,
1995) and also as unusual histologic finding with
HGF (Gunhan et al., 1995).This possible
implications between the pathogenesis of GO and
tumorigenesis suggested the aim of the present
study which was the examination of the expression
of a tumor-related marker, Ki-67, in hyperplastic
gingival tissues induced by cyclosporine and
phenytoin as well as cases of HGF and compare
them to healthy control tissues.
Currently, more than 15 drugs have been
identified as possible causative agents of gingival
overgrowth. However, phenytoin and cyclosporine
are more commonly involved (Lin et al., 2007;
Silverstein et al., 1997). One property that is
common for these two different classes of drugs is
that they directly affect cellular calcium
metabolism. Since cellular production of
collagenase is modulated by calcium influx,
fibroblasts from patients treated with these drugs
may produce an inactive form of collagenase, being
responsible for an increase in the extracellular
matrix (Brunet et al., 1996). Combined with this
reduction in extracellular matrix degradation;
enhanced proliferation of keratinocytes and/or
resident fibroblasts were reported (Saito et al.,
1999; Nurmenniemi et al., 2001). These previous
findings align with the histopathologic changes and
the significant increase in the optical density of Ki-
67 staining reported in the current study within the
epithelium and corium of both cyclosporine and
phenytoin induced GO groups when compared to
the control tissue samples.
Nurmenniemi et al., (2001) also reported a
significant increase in numbers of Ki-67–labeled
cells in patients with CGO compared to healthy
controls. Saito et al., (1999) found that mean rates
of Ki-67–positive cells in PGO were significantly
higher as well than healthy tissues. These conflicts
with a previous study reported that the acanthosis
observed in cyclosporine-treated patients is not
caused by enhanced keratinocytes proliferation but
rather by prolonged cell life caused by an
antiapoptotic effect of cyclosporine (Niimi et al.,
1990). Bulut et al.,(2004) revealed that epithelial
proliferation rates may be unchanged in renal
transplant patients with CGO when compared to
healthy controls.
As for HGF, most attention has been focused
on the proliferative potential of mesenchymal
fibroblasts. In the present study, both epithelium
and connective tissue cells were studied and no
significant difference was found in the proliferative
potential of epithelium and connective tissue as
reflected by Ki-67 optical density which was higher
than control tissues and comparable to that of PGO
group. In corroboration, a study with 12 different
cell lines from patients of a Brazilian HGF family
demonstrated a significantly higher proliferation
rate of HGF fibroblasts compared to fibroblasts
from normal gingivae (de Andrade et al., 2001). On
the other hand, Saygun et al.,(2003) suggested that
the underlying mechanism of HGF are not involved
with increased cellular proliferation and that lack of
proliferation is caused by unfavorable cellular
environment lacking key nutrients caused by
excessive extracellular matrix deposition.
Findings from the current study confirmed
that increased cell division and proliferation may
have a role in the pathogenesis of drug-induced
gingival overgrowth as well as HGF, however;
several factors, including age, genetic
predisposition, pharmacokinetic variables and
plaque-induced inflammatory changes are believed
to be important in the onset and severity of gingival
overgrowth. Accordingly, further studies with
larger sample size will provide more conclusive
data on the possible role of enhanced proliferative
activity of cells in the pathogenesis of gingival
overgrowth.
Acknowledgements
Histopathologic and immunohistochemical
procedures and evaluation were performed at the
Department of Oral Pathology, Faculty of Oral and
Dental Medicine, Cairo University.
Corresponding author
Eman Y. El-Firt
Departement of Oral Medicine and Periodontology,
Faculty of Oral and Dental Medicine, Cairo
University, Cairo, Egypt
elfirt@yahoo.com
References
Brunet, L.; Miranda, J.; Farré, M.; Berini, L. and
Mendieta, C. (1996): Gingival enlargement
induced by drugs. Drug Saf.; 15:219-231.

Life Science Journal, 2011;8(X) http://www.lifesciencesite.com
http://www.sciencepub.net/life lifesciencej@gmail.com
226
Buduneli, N.; Buduneli, E.; Cxınar, S.; Lappin, D.
and Kinane, D.F.(2007): Immunohistochemical
evaluation of Ki-67 expression and apoptosis in
cyclosporin A-induced gingival overgrowth. J
Periodontol.; 78:282-289.
Bulut, Ö.E. ; Sökmensüer, L.K.; Bulut, S.; Tasman,
F. and Müftüoǧlu S.(2004):
Immunohistochemical study of cyclosporin-
induced gingival overgrowth in renal transplant
recipients. J Periodontol.; 75:1655-1662.
Coletta, R.D. and Graner, E.(2006): Hereditary
Gingival Fibromatosis: A Systematic Review. J
Periodontol.; 77:753-764.
de Andrade, C.R.; Cotrin, P.; Graner, E.; Almeida,
O.P.; Sauk, J.J. and Coletta, R.D.(2001):
Transforming growth factor beta-1autocrine
stimulation regulates fibroblast proliferation in
hereditary gingival fibromatosis. J Periodontol.;
72:1726-1733.
Gunhan, O.; Gardner, D.G.; Bostanci, H. and
Gunhan, M.(1995): Familial gingival fibromatosis
with unusual histologic findings. J Periodontol.;
66:1008-1011.
Lin, K.; Guilhoto, L.M.F. and Yacubian,
E.M.T.(2007): Drug-induced gingival
enlargement – Part II. Antiepileptic Drugs: not
only phenytoin is involved. J Epilepsy Clin
Neurophysiol.; 13:83-88.
Mariani, G.; Calastrini, C.; Carinci, F.; Marzola, R.
and Calura, G.(1993): Ultrastructural features of
cyclosporin A-induced gingival hyperplasia. J
Periodontol.; 64:1092-1097.
Martelli-Junior, H.; Lemos, D.P.; Silva, C.O.;
Graner, E. and Coletta, R.D.(2005): Hereditary
gingival fibromatosis: Report of a five-generation
family with cellular proliferation analysis. J
Periodontol.; 76:2299-2305.
Martelli-Junior, H.B.G.; Graner, E.; Bozzo, L. and
Coletta, R.D. (2000): Microscopic and
proliferative comparison of gingival fibroblasts
from patients with normal gingiva and with
hereditary gingival fibromatosis. Pesqui Odontol
Bras.; 14:123-129.
McLoughlin, P.; Newman, L. and Brown, A.
(1995):Oral squamous cell carcinoma arising in
phenytoin-induced hyperplasia. Br Dent J.;
178:183-4.
Niimi, A.; Tohnai, I.; Kaneda, T.; Takeuchi, M. and
Nagura, M. (1990): Immunohistochemical
analysis of effect of cyclosporine-A on gingival
epithelium. J Oral Pathol Med.; 19:397-403.
Nurmenniemi, P.K.; Pernu, H.E. and Knuuttila,
M.L.E.(2001): Mitotic activity of keratinocytes in
nifedipine- and immunosuppressive medication-
induced gingival overgrowth. J Periodontol.;
72:167-173.
Rateitschak-Plüss, E.M.; Hefti, A.; Lörtscher, R.
and Theil G.(1983): Initial observation that
cyclosporin-A induces gingival enlargement in
man. J Clin Periodontol.; 10:237-246.
Saito, K.; Mori, S.; Tanda, N. and Sakamoto,
S.(1999): Expression of p53 protein and Ki-67
antigen in gingival hyperplasia induced by
nifedipine and phenytoin. J Periodontol.; 70:581-
586.
Saygun, I.; Ozdemir, A.; Gunhan, O.; Aydintug,
Y.S. and Karslioglu, Y.(2003): Hereditary
gingival fibromatosis and expression of Ki-67
antigen: A case report. J Periodontol.; 74:873-
878.
Schlüter, C.; Duchrow, M.; Wohlenberg, C.; et al.
(1993): The cell proliferation-associated antigen
of antibody Ki-67: a very large, ubiquitous
nuclear protein with numerous repeated elements,
representing a new kind of cell cycle-maintaining
proteins. J. Cell Biol.; 123:513-22.
Silverstein, L.H.; Garnick, J.J.; Szikman, M. and
Singh, B.(1997): Medication induced gingival
enlargement: a clinical review. Gen Dent.;45:371-
376.
Singer, S.L.; Goldblatt, J.; Hallam, L.A. and
Winters, J.C.(1993): Hereditary gingival
fibromatosis with a recessive mode of inheritance.
Case reports. Aust Dent J.; 38:427-432.
Vardar, S.; Baylas, H.; Zihniog˘lu, F.; Buduneli, N.;
Emingil, G. and Atilla, G.(2005): Gingival tissue
proteoglycan and chondroitin sulphate levels in
cyclosporin A-induced gingival overgrowth and
the effects of initial periodontal treatment. J Clin
Periodontol.; 32:634-639.
Varga, E. and Tyldesley, W.R.(1991): Carcinoma
arising in cyclosporin-induced gingival
hyperplasia. Br Dent J.; 171:26-27.
Zoeller, J.; Flentje, M.; Sinn, P. and Born,
I.A.(1994): Evaluation of AgNOR and Ki-67
antigen as cell kinetic parameters in oral
dysplasias and carcinomas. Anal Cell Pathol.;
7:77-88.
10/24/2011