103

Journal of Oral Science, Vol. 44, No. 2, 103-108, 2002

Original

Behavior of HO-1-N-1, buccal mucosa carcinoma derived cells, on [Ca2+]i responses to stimulants

Takafumi Arai§, Hiroko Matsumoto§, Yoshiaki Akimoto†,

Hitoshi Nishimura†, Makiko Ono†and Akira Fujii§

§Department of Pharmacology and†Second Department of Oral Surgery,

Nihon University School of Dentistry at Matsudo, Chiba 271-8587

(Received 27 February and accepted 14 June 2002)

Abstract: Buccal mucosa carcinoma-derived cell

line, HO-1-N-1, epithelial-like cells, was obtained in order to investigate the characteristics of oral cancer cells and examine the [Ca2+]i responses to stimulants,

such as bradykinin (BK), histamine (HIST), thapsigargin (TG), epidermal growth factor (EGF)

and transforming growth factor a (TGF a ).

Intracellular Ca2+ influx was observed by all stimulants that enhanced the [Ca2+]i response. However,

intracellular Ca2+ release was not observed in response

to growth factors. The [Ca2+]i response of BK (100 nM) was inhibited by 10 iu M of the BKB2 antagonist, D-Arg-[Hyp3, Thi5, 8, D-Phe7]-BK, and HIST (1 mM) was

completely inhibited by 100nM of the Hi antagonist,

(+)-chlorpheniramine, in the presence and absence of extracellular Ca2+ (1.5mM). (J. Oral Sci.44, 103-108,

2002)

Key words: HO-1-N-1 cell; [Ca2+]i; bradykinin; histamine.

Introduction

Calcium signaling transduction can regulate such diverse

cellular processes as fertilization, cell growth, nerve cell transduction, smooth muscle concentration and neuronal signaling. Berridge (1) described the cell transformation

in the following manner: Cancer cells are characterized

by uncontrolled and unlimited growth. It becomes tumorigenic as a result of multiple independent steps that

subvert the normal growth control mechanism. Some of these steps have been linked with mutations that either

activate proto-oncogenes such as ras and myc, or remove the inhibitory action of tumor-suppressor genes such as

Rb and p53. With regard to calcium, cell transformation leads to a much reduced calcium requirement for growth

(1,2). Since intracellular calcium has an important role in

tumor transformation (3-6), we examined the intracellular Ca2+ concentration, [Ca2+]i, responses to stimulants, such

as bradykinin (BK), histamine (HIST), thapsigargin (TG), epidermal growth factor (EGF), transforming growth factor a (TGF a ), thrombin, interleukin-1 and PGEs (7), in

order to reveal the characteristics of HO-1-N-1, buccal mucosa carcinoma-derived cell line.

Materials and Methods

Chemicals and reagents

BK, ionomycin, HIST, EGTA, TG, EGF, TGF a ,

thrombin, IL-1 a , IL-1 ƒÀ , PGE1 and PGE2 were purchased

from Sigma Chemical Co., USA. The BKB 1 antagonist,

Des-Arg9-[Leu8]-BK, and the BKB2 antagonist, D-Arg-

[Hyp3, Thi5, 8, D-Phe7]-BK, were obtained from Peptide

Institute, Japan. Fura-2/AM was purchased from Dojin

Laboratories, Japan. All the chemicals and reagents for

tissue culture were purchased from Gibco Laboratories,

USA.

Cells

The buccal mucosa carcinoma-derived cell line, HO-1-

N-1, epithelial-like cells from the Japan Health Science

Correspondence to Dr. Akira Fujii, Department of Pharmacology, Nihon University School of Dentistry at Matsudo, 2-870-1 Sakaecho-Nishi, Matsudo, Chiba 271-8587, Japan Tel: +81-47-360-9344 Fax: +81-47-360-9348 E-mail: afujii@mascat.nihon-u.ac.jp

104

Foundation, was incubated in an atmosphere of 5% CO2 - 95% air at 37°C in a medium of DMEM: RPMI 1640(1:1

w:w) supplemented with 10% FCS and 100 is g/ml kanamycin.

Determination of [Ca2+]i

The determination of [Ca2+]i was carried out by the

procedure previously reported (7,8). Thus, HO-1-N-1

was harvested with 0.25% trypsin-0 .02% EDTA. Fura-2

/AM (50 ,ƒÊ g) was dissolved in 25 ,u1 of dimethylsulphoxide

and 187,ƒÊ l of fetal calf serum (FCS) was added . The

mixture was added to 6 ml of physiological salt solution

(PSS) supplemented with 10% of FCS and then sonicated

with an Ultrasonic Processor (50V , 30s, VP-5, Taitech,

Japan). Cell suspension (4 ml in PSS supplemented with

10% FCS) was added to the sonicated solution to make a

final Fura-2/AM concentration of 5 ,u M containing

approximately 4x 106 cells/ml. Cells were loaded with

Fura-2/AM at 37•Ž for 30 min, and then washed three times

with PSS. The Fura-2 loaded cell suspension (3.6•~105

cells/nil) was kept on ice. A portion (2 ml) of the cell

suspension was placed in a cuvette, preincubated for 4 min

at 37°C, and then fluorescence emission (at 500 nm) was

measured at two different excitation wavelengths, 340

and 380nm, at 37°C using a CAF-110 Ca2A- Analyzer

(JASCO Corporation, Japan). For the measurement in the

absence of Ca2+ in the extracellular fluid, PSS without Ca2+

substituted with 0.5mM EGTA was used in the same

manner as described previously (7,8).

[Ca2+]i responses to BK, HIST, TG, EGF, TGF a ,

thrombin, IL-1 ƒ¿ , IL-1 ƒÀ , PGE1 and PGE2

[Ca2+]i responses to different concentrations of BK (1

nM to 10,ƒÊ M) and HIST (10,ƒÊ M to 1mM) were

determined in the presence of 1.5 mM Ca2+. [Ca2+]i

responses to 100,u M BK, 1 mM HIST, 1,u M TG , 1,u g/ml

EGF, 500 ng/ml TGF ƒ¿ , 1 U/ml thrombin, 500 pg/ml IL-

1 ƒ¿ , 500 pg/ml IL-1 ƒÀ , 500 ng/ml PGE1 and 500 ng/ml

PGE2 were also determined. Ca2+ influx was determined

in the same manner as described previously (7 , 8). Thus,

after pretreatment with either BK, HIST, TG , EGF, TGF

a , thrombin, IL-1 ƒ¿ , IL-1 /3 , PGE1, or PGE2 for 2 min,

in Ca2+ free medium (omission of CaC12 addition in PSS)

substituted with 0.5 mM EGTA, Ca2+ (final concentration

of 1.5 mM) was re-added to the suspension medium . The

increase of [Ca2+]i was regarded as the Ca2+ influx .

Effect of bradykinin and histamine antagonists

Cells were pre-treated with different concentrations

(1 ,ƒÊ M or 10 ƒÊ M) of BK antagonists (BKB1 antagonist,

Des-Arg9-[Leu8]-BK, and BKB2 antagonist, D-Arg-

[Hyp3,Thi5, 8,D- Phe7]-BK ) or different concentrations (1

nM or 100 i M) of histamine antagonists (H1 antagonist,

(+)-chlorpheniramine (CPM), and H2 antagonist,

cimetideine (CIMN)) for 2 min after incubation at 37°C

for 4 min and then 100 nM BK or 1 mM HIST were

added, respectively, to clarify whether these receptors

were responsible for the [Ca2+]i responses .

Results

The dose-response curves of [Ca2+]i produced by BK

and HIST are shown in Fig. 1A and Fig. 1B, respectively .

[Ca2+]i was increased dose-dependently from 10-9 M to

10-6 M by the BK treatments and then reached a plateau .

HIST from 10-5M to 10-3M also produced transient dose-

dependent [Ca2+]i increases.

The first transient increases of [Ca2+]i produced by 100

nM BK, 1 mM HIST, 1,ƒÊ M TG, 1,ƒÊ g/ml EGF , 500 ng/ml

TGF ƒ¿ , 1 U/ml thrombin, 500 pg/ml IL-1 ƒ¿ , 500 pg/ml

IL-1 ƒÀ , 500 pg/ml PGE1 and 500 pg/ml PGE2 are

Fig. 1 Dose-response of [Ca2+]i produced by BK (A) and HIST (B).

105

summarized in Table 1. BK, HIST, TG, EGF and TGF a

stimulated a [Ca21i response, but thrombin, IL-1 and PGs did not under the present experimental conditions.

Figs. 2 and 3 show the effect of BKB1 and BKB2

antagonists against the [Ca2+]i response elicited by BK in

the presence or absence of extracellular Ca2+, respectively.

The BKB1 antagonist did not have an effect on the [Ca2+]i

response, however the BKB2 antagonist reduced the [Ca2+]i

response by 0.8% for the 1 u M and 54.9% for the 10 ti

M.

107

of the EGF receptor triggers a cascade of events that are thought to be responsible for the initiation of DNA synthesis

and cell cycle progression. The EGF receptor mediates

the transmembrane signaling of at least two other EGF-like molecules, TGF a and vaccina virus growth factor (14).

Thus, EGF and TGF a bound to the EGF receptor, but did not accelerate PLC 7, and induced Ca2+ influx (Table 1).

TG is an inhibitor of endoplasmic/sarcoplasmic Ca2+- ATPase and binds with high affinity to Ca2+ pumps resulting

in the inhibition of Ca2+ influx into the Calf store, the ER

(17). TG also activates TG-induced emptying diffusible messengers and accelerates Ca2+ influx through the plasma membrane. Results shown in Table 1 indicate that HO-1-

N-1 cells possess TG sensitive Ca2+-ATPase.

Many mammalian cells possess the protease-activated

receptor (PAR), which is activated by protease, such as thrombin and trypsin, and by synthetic peptides

corresponding to the new tethered ligand N-terminus of PAR created by receptor cleavage (18,19). Thrombin

activates PAR-1, PAR-3 and PAR-4, resulting in a transient

[Ca2+]i increase (20). Since HO-1-N-1 did not produce any transient [Ca2+]i increase, the cell might not possess PAR-

CaT-like, a novel calcium-sensitive channel, correlates with the malignancy of prostate cancer.

J Biol Chem 276, 19461-19468

5. Chung KC, Sung JY, Ahn W, Rhim H, Oh TH, Lee MG, Ahn YS (2001) Intracellular calcium mobilization induces immediate early gene pip92

via Src and mitogen-activated protein kinase in immortalized hippocampal cells. J Biol Chem 276,

2132 - 2138 6. Wartenberg M, Diedershagen H, Hescheler J, Sauer

H (1999) Growth stimulation versus induction of cell

quiescence by hydrogen peroxide in prostate tumor spheroids in encoded by the dulation of the Ca2+

response. J Biol Chem 274, 27759-27767

7. Fujii A, Matsumoto H, Hashimoto T, Akimoto Y

(1995) Effect of bradykinin on cytosolic calcium response in cultured gingival fibroblasts derived from nifedipine responders and non-responders.

Cell Pharmacol 2, 171-176 8. Fujii A, Matsumoto H, Hashimoto T, Akimoto Y

(1995) Tenidap, an anti-inflammatory agent, discharges intracellular Ca++ store and inhibits Ca"

Table 1 Comparison of the first rapid rise of [Ca2+]i by stimulants

106

Figs. 4 and 5 show the effect of Hi and H2 antagonists against the [Ca21i response elicited by HIST . CPM, an H1 antagonist, completely inhibited the [Ca2±]i response in the presence or absence of extracellular Ca2+.

Discussion Leukoplakia is defined as a white patch or plaque that

cannot be characterized clinically or pathologically as any

other disease such as oral lichen planus , candidiasis, white sponge nevus, and others (9) . It should be regarded as a

potentially premalignant lesion due to dyskeratosis and loss of the epithelial basement lesion . The malignant transformation rate of leukoplakia is reported to be 3 to 30% (10). As a first step , we obtained buccal mucosa carcinoma-derived cell line, HO-1-N-1, epithelial-like cell from the Japan Health Science Foundation , in order to investigate the nature of transformed epithelial cells .

In non-excitable cells, such as epithelial cells and fibroblasts, the [Ca2li response is predominantly controlled by the phosphatidylinositol pathway . The production of

IP3 is mediated by phospholipase C (PLC) from

phoshatidylinositol - 4,5 - bisphosphate (PIP2). There are

two types of PLC, PLCƒÀ and PLCƒÁ , which are activated

by a G protein-coupled receptor class of seven

transmembrane-spanning receptors (GCRs) and the receptor

tyrosine kinase (RTKs), respectively (1) . The IP3 receptor

spans the endoplasmic reticulum (ER) and triggers the

release of Ca2+ from the ER (Ca2+ store) (1-8, 11-14). Thus,

[Ca2+]i is increased by the activation of PLC through these

two receptors. The presence of these two PLCs has been

reported in epithelial cells and their transformed cells

(15,16).

In this study, we demonstrated that BK and HIST

increased transient [Ca2+]i in Fura-2/AM loaded HO -1-N-

1, indicating these cells possess BKB2 and H1 receptors,

respectively, resulting in activation of PLC /9. Both HIST

and BK are quite common in the process of inflammation,

and the epithelial cells might be responsible, to some

extent, in the first step of protection . EGF and TGFƒ¿ did

not enhance [Ca2+]i , but produced Ca2+ influx. Activation

Fig. 4 The [Ca2+]i response to 1 mM HIST was inhibited by 100 nM CPM in the presence of extracellular Ca2+.

Fig. 5 The [Ca2+]i response to 1 mM HIST was inhibited by

100 nM CPM in the absence of extracellular Ca2+. The omission of Ca2+ addition in the PSS substituted

with 0.5 mM of EGTA was regarded as the absence of extracellular Ca2+.

107

of the EGF receptor triggers a cascade of events that are

thought to be responsible for the initiation of DNA synthesis and cell cycle progression. The EGF receptor mediates

the transmembrane signaling of at least two other EGF-like molecules, TGF a and vaccina virus growth factor (14). Thus, EGF and TGF a bound to the EGF receptor, but did

not accelerate PLC y , and induced Ca2+ influx (Table 1). TG is an inhibitor of endoplasmic/sarcoplasmic Ca2+-

ATPase and binds with high affinity to Ca2+ pumps resulting in the inhibition of Ca2+ influx into the Ca2+ store, the ER

(17). TG also activates TG-induced emptying diffusible messengers and accelerates Ca2+ influx through the plasma

membrane. Results shown in Table 1 indicate that HO-1-N-1 cells possess TG sensitive Ca2+-ATPase.

Many mammalian cells possess the protease-activated receptor (PAR), which is activated by protease, such as

thrombin and trypsin, and by synthetic peptides corresponding to the new tethered ligand N-terminus of

PAR created by receptor cleavage (18,19). Thrombin activates PAR-1, PAR-3 and PAR-4, resulting in a transient

[Ca2+] increase (20). Since HO-1-N-1 did not produce any transient [Ca21i increase, the cell might not possess PAR-1, PAR-3, nor PAR-4 under the present experimental

conditions. Lourbakos et al. (21) demonstrated that human

oral epithelial cells (KB cells) were found to express PAR-

1, PAR-2, and PAR-3 on their surface, but not PAR-4. Thus, further study might be necessary to clarify the presence

of PARs on the surface of HO-1-N-1.

Acknowledgments

This work was supported in part by a Grant from the Ministry of Education, Culture, Sports, Science and

Technology to promote 1998-Multidisciplinary Research Projects (in 1998-2002)

References

1. Berridge MJ(1993) Inositol trisphosphate and calcium signalling. Nature 361, 315-325

2. Yoshida T, Takahashi Y, Takashima S (1993) Effect of low extracellular Ca2+ on growth, spreading area, cytoplasmic Ca2+ concentration, and intracellular pH

in normal and transformed human fibroblasts. J Cell Physol 154, 301-309

3. Kawanabe Y, Hashimoto N, Masaki T (2001) Ca2+ influx through nonselective cation channels plays

an essential role in endothelin-induced mitogenesis in C6 glioma cells. Neuropharmacology 41, 331-

340 4. Wissenbach U, Niemeyer BA, Fixemer T,

Schneidewind A, Trost C, Cavalie A, Reus K, Meese E, Bonkhoff H, Flockerzi V (2001) Expression of

CaT-like, a novel calcium-sensitive channel, correlates with the malignancy of prostate cancer.

J Biol Chem 276, 19461-19468 5. Chung KC, Sung JY, Ahn W, Rhim H, Oh TH, Lee

MG, Ahn YS (2001) Intracellular calcium

mobilization induces immediate early gene pip92 via Src and mitogen-activated protein kinase in

immortalized hippocampal cells. J Biol Chem 276, 2132 - 2138

6. Wartenberg M, Diedershagen H, Hescheler J, Sauer H (1999) Growth stimulation versus induction of cell

quiescence by hydrogen peroxide in prostate tumor spheroids in encoded by the dulation of the Ca2+ response. J Biol Chem 274, 27759-27767

7. Fujii A, Matsumoto H, Hashimoto T, Akimoto Y

(1995) Effect of bradykinin on cytosolic calcium response in cultured gingival fibroblasts derived from nifedipine responders and non-responders.

Cell Pharmacol 2, 171-176

8. Fujii A, Matsumoto H, Hashimoto T, Akimoto Y

(1995) Tenidap, an anti-inflammatory agent, discharges intracellular Ca++ store and inhibits Ca"

influx in cultured human gingival fibroblasts. J Pharmacol Exp Ther 275, 1447-1452

9. Krmer IR, Lucas RB, Pindborg JI. Sobin LH (1978)

Definition of leukoplakia and related lesions: an aid to studies on oral precancer. Oral Surg Oral

Med Oral Pathol 46, 518-539 10. Matsushima T, Hirata S, Ando R, Masuda S,

Hirakawa K, Yajin K, Murakami I (1997) The relationship between oral leukoplakia and malignant

potentiality : clinicopathological study and analysis of the p53 gene mutation. Koku Intoka. 9, 453-458

(in Japanese) 11. Ghosh TK, Bian J, Short AD, Rybak SL, Gill DL

(1991) Persistent intracellular calcium pool depletion by thapsigargin and its influence on cell growth. J Biol Chem 266, 24690-24697

12. Bian J, Ghosh TK, Wang J-C, Gill DL (1991) Identification of intracellular calcium pools. Selective modification by thapsigargin. J Biol Chem 266,

8801-8806 13. Sagara Y, Inesi G (1991) Inhibition of the

sarcoplasmic reticulum Ca2+ transport ATPase by thapsigargin at subnanomolar concentrations. J Biol

Chem 266, 13503-13506 14. Clapham DE (1995) Calcium signaling. Cell 80, 259-

268 15. Knorr M, Steuhl KP, Dartsch P, Thiel HJ (1990)

Activation of phosphatidylinositol metabolism of

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108

factor. Fortschr Ophthalmol 87, 649-652 (in German)

16. Oakes SG, Iaizzo PA, Richelson E, Powis G (1988) Histamin-induced intracellular free Ca++, inositol

phosphates and electrical changes in murine N1E-115 neuroblastoma cells. J Pharmacol Exp Ther

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Dawson AP (1990) Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific

inhibition of the endoplasmic reticulum Ca2+- ATPase. Proc Natl Acad Sci USA 87, 2466-2470

18. Vu TK, Hung DT, Wheaton VI, Coughlin SR (1991)

Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor

activation. Cell 64, 1057-1068 19. Ishihara H, Connolly AJ, Zeng D, Kahn ML, Zheng

YW, Timmons C, Tram T, Coughlin SR (1997)

Protease-activated receptor 3 is a second thrombin receptor in humans. Nature 386, 502-506

20. Chang M-E, Chan C-P, Wu H-L, Chen R-S, Lan W-H, Chen Y-J, Jeng J-H (2001) Thrombin-stimulated

growth, clustering, and collagen lattice contraction of human gingival fibroblasts is associated with its

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Andrade-Gordom P, Santulli R, Mackie EJ, Pike BN

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5121-5130

109

Journal of Oral Science, Vol. 44, No. 2, 109-111, 2002

Case report

Malignant potential of the reticular form of oral lichen planus

over a 25-year observation period in 55 patients from Slovenia

Matj aZ Rode§and Mirela Kogoj -Rode†

§Department of Oral Pathology, Medical Centre, Lj ublj ana-Slovenia,

†Department of Stomatology , University Clinical Centre, Lj ub lj ana|Slovenia

(Received 28 August 2001 and 23 May 2002)

Abstract: A retrospective evaluation of the reticular form of oral lichen planus was made on the basis of

clinical and histopathological observations in 55

patients. Patients were re-examined once every six months over a 25-year period. Biopsies were taken

from all lesions. No cases of complete remission and no cases of malignant transformation were recorded. Our findings suggest that the reticular form of oral lichen

planus is not a precancerous lesion. (J. Oral Sci. 44, 109- 111, 2002)

Key words: oral cavity; precancerous lesions; oral lichen planus.

Introduction

Lichen planus ( LP ) is a relatively common disorder

which affects 0.5% to 1.9% of the population. Approximately 20% of patients in a referral oral surgery

practice were diagnosed as having oral lichen planus (OLP) (1,2).

OLP is a chronic inflammatory disease of the oral

mucous membranes. It shows a variety of clinical appearances: reticular, papular, plaque-like, erosive,

ulcerative, bullous and atrophic (3-6). Reticular OLP is

generally characterized by lesions consisting of radiating white or grey papules in a linear, annular or retiform

arrangement, forming typical reticular patches, rings and streaks on the oral mucosa. The reticular form is the most common and is generally asymptomatic, while the

ulcerative and bullous forms are frequently associated

with pain. Compared with skin lesions, mucosal LP is more chronic in nature with less than 20% of cases showing

complete remission (3). OLP is histopathologically well defined, characterized

by a T-cell-dominated infiltrate next to the basal cell layer of the epithelium, and by epithelial basal cell destruction and thickening or disruption of the basement membrane

(2,6). However the aetiology of OLP is very complex and the

pathological process remains obscure (2,7). Results of numerous investigations indicate that cell-mediated

mechanisms are involved in the initiation and progression of the lesions (2-5). It is reasonable to assume that OLP

is a localized autoimmune disease. Walsh et al. have suggested that modified keratinocyte surface antigens are

the target for the cytotoxic cell response, whereas mast cells, antigen-presenting Langerhans cells and cytokines

produced by lymphocytes and keratinocytes play a key role in the evolving lesion (7).

The possibility of malignant transformation of OLP is

still questionable, and continues to be discussed in the literature (8-11). A number of mainly retrospective studies from several countries have shown only a minimal risk of

malignant transformation (12-15). Silverman et al.(14) reported malignant transformation in 1.2% of cases of

OLP, and Holmstrup et al. (16) in 1.5% of cases.

Materials and Methods

Clinical data, including age, gender, previous medical history, medications, therapeutic protocols and malignant

association from 55 patients with the reticular form of oral lichen planus were obtained from a previous survey

conducted by the Department of Clinical Oral Pathology at the Medical Centre in Ljubljana. The patients had no

Correspondence to Dr. Matja2 Rode, Department of Oral Pathology, Medical Centre, Ljubljana-Slovenia, Bratov Ucakar 16, 1000 Ljubljana, SloveniaTel : 386-1-507-48-30E-mail : matjaz.rode@siol.net

110

symptoms and had been referred to the specialist when their

dentist had discovered the lesions. All patients were

questioned about their previous medical and dental histories, as well as the history of the current OLP lesions. The

patients had been studied since 1970 and were followed through to 1994. The mean age at initial presentation was

58.3 years with a standard deviation of 5.1 years. The age

range was from 41 to 64 years. Of the 55 patients 44, (80%)

were women. Oral examinations of all lesions were

performed by three investigators double checking each other in daylight and using a mouth mirror. Oral reticular lichen

planus was diagnosed and registered on a clinical basis. Scores of 0 to 3 were recorded according to the following

criteria:

Score 0 = no lesions, normal mucosa

Score 1 = white striae, less than 1 cm2

Score 2 = white striae, more than 1 cm2

Score 3 = white striae, more than 2 cm2..

Biopsies under local anaesthesia were taken of oral

mucosal lesions of all patients. The samples were fixed in

10% formaldehyde solution for routine histopathology. The

diagnosis of OLP was confirmed histologically at the Institute of Pathology; Medical Faculty of Ljubljana.

No patient had characteristic skin lesions. Patients with

lichenoid lesions close to amalgam dental fillings and

patients with oral lichenoid drug eruptions were not included in the group under observation .

The patients were re-examined once every six months and lesions showing clinical changes were registered .

Biopsies were repeated after 10 years of observation in 9

patients with a progredient OLP. Patients received no treatment during the years under observation.

Results 1. Location of the lesions

The most common lesion site was the buccal mucosa, followed in decreasing order of incidence by the retromolar

pads, the tongue and the floor of the mouth and the gingiva. Multiple lesion sites occurred frequently (Table 1).

2. Lesion evolution

Disease remission was noted in 14 patients after initial

presentation. A more pronounced expression of the lesion was noted in 9 patients and an unchanged situation was observed in 32 patients over a 25-year period after initial

observation (Table 2). Transformation into other forms of OLP was not observed.

3. Histopathological findings All tissue speciments showed similar histological

Table 1 Lesion sites in 55 patients with oral lichen planus

Table 2 Evolution of lesions during a 25-year observation-

period

Fig. 1 Histopathology of reticular oral lichen planus:

parakeratosis, vacuolar degeneration of basal cells, typical lymphocytic infiltrate. (HE, X 195)

111

abnormalities. The epithelium of the oral mucosa was hyperplastic with acanthotic projections, and hyperplasia

was seen in the spinous layer. The epithelium was covered by a parakeratotic layer. Liquefaction of the basal cell

layer was observed focally, accompanied by marked mononuclear infiltrate at the dermoepidermal junction. No

atypia of the epithelial cells was found. Histopathological findings after 10 years of observations were similar to

previous findings. No dysplasia was found (Fig. 1).

4. Malignant transformation No malignant alteration was detected in any of the 55

patients.

Discussion

The data presented are consistent with data from previous

studies with regard to location and disease chronicity

(1,5,17) . Disease remission was observed in some patients without treatment. Patients were predominantly female. The procedure of routine biopsy in the diagnosis of OLP

is controversial (3). In our study biopsies were routinely taken. All tissue specimens showed similar histological

abnormalities. No atypia of the epithelial cells was found. We recorded no cases of malignant transformation in 55

patients followed up for 25 years. Our results confirm the findings of Brown et al. (1) and do not support the

observations of Silverman et al. (17) who reported malignant transformation in one case of reticular OLP. The

results of Holmstrup et al. (16) who reported that reticular OLP is related to oral cancer could not be confirmed

either. We recorded disease remission without therapy in 14

patients and intensification of mucosal striae in 9 patients. Our study does not support the observations of Brown et

al. (1) who suggested a complete remission. Malignant transformation of any form of oral lichen

planus cannot be discounted. It would therefore seem

prudent clinical practice always to confirm the clinical diagnosis of OLP histologically, and to have patients followed up regularly.

It is suggested that a biopsy should be performed in all

instances of OLP and that the patients should be followed up regularly.

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