Zhong et al.

Int Chin J Dent 2004; 4: 27-33.

Expression of telomerase and p53 in ameloblastoma of the jaw
Ming Zhong, DDS, MS,a Luhuang Zhang, DDS, MD,a Jie Wang, DDS, MD,a Bo
Zhang, DDS, PhD,b and Lin Hou, DDSbab

Department of Pathology, College of Stomatology, China Medical University, Shenyang, andDepartment of Pathology, Medical College, Peking University, Beijing, P. R. China

Purpose: This study was aimed to detect the expression of p53 protein, hTR, and hTERT (telomerase
reverse transcriptase, TERT) mRNA in ameloblastoma (AB) of the jaw, and to investigate its correlation
with the clinical and biological features of AB.
Materials and Methods: In situ hybridization and immunohistochemical assay were used to detect theexpression of HTR and hTERT mRNA in 54 cases of AB. Meanwhile, S-P method was used to determine
the expression of p53 protein in 49 cases of ABs. Specimens from 25 cases of odontogenic keratocyst(OKC) and seven cases of normal oral mucosa were used as control. Chi square test and Kendall Pearson
correlation analysis were used to determine the relationship between gene expression and clinical featuresof AB.
Results: The positive ratio of hTR, hTERT mRNA was 2/7 and 1/7 in normal oral mucosa, respectively;
81.2% (13/16) and 87.4% (14/16) in odontogenic keratocyst (OKC), 94.4% (51/54) and 94.4% (51/54) in
AB, respectively. There were significant differences among these three groups (p<0.001). The hTR wasstrongly correlated with hTERT mRNA (rs=1.000, p<0.001). The p53 protein was expressed in nucleolusof 87.5% (42/49) AB, 44% (11/25) OKC, 33.3% (1/3) normal oral mucosa, respectively. A strong
correlation between hTERT mRNA and p53 protein was found (rs=0.754, p<0.001). An intermediate
positive correlation between hTR mRNA and p53 protein was noted (rs=0.536, p<0.001).
Conclusion: The positive ratios of hTR and hTERT were significantly higher than p53 protein in AB.
Telomerase activity and p53 expression play an important role in genesis and development of AB.
(Int Chin J Dent 2004; 4: 27-33.)
Clinical Significance: The study investigated that the high expression of hTR and hTERT was closely
related to the clinical biological behavior of AB and regulated by p53 protein, which provided a significant
proof for looking for a new target to therapy AB by gene.
Key Words: ameloblastoma, hTERT, hTR, odontogenic keratocyst, p53 protein, telomerase.

Introduction
The activated telomerase was first found in human cervical cancer cells by Morin in 1989. In 1994, Kim
et al.1 described the highly sensitive telomerase repeat amplification procedure (TRAP) based on PCR for
the first time, which enables the detection of the activity of telomerase. Currently TRAP was extensively
applied in studies of various tumors and carcinogenesis. The expression of the activity of telomerase
involves a complex mechanism and regulated by multitudinous factors. However, the expression of hTR,
hTERT and p53 protein, and their relations in ameloblastoma (AB) and odontogenic keratocyst (OKC) of
the jaw were not clear until now. This study was aimed to detect the expression of p53 protein, hTR, andhTERT mRNA in ameloblastoma (AB) of the jaw, and to investigate its correlation with the clinicalbiological characteristics of AB.

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Int Chin J Dent 2004; 4: 27-33.

Materials and Methods
Tissue Specimens
The tissue specimens were filed paraffin-embedded from the Pathological Department of the First
Affiliated Clinical College of China Medical University between the year of 1996 and 2000, which
included 54 ameloblastomas of the jaw. Among them, 31 were primary, 19 were recurrent, and four werewith malignant transformation. There were 25 males and 29 females, their age ranged from nine to 69
years with an average of 33.4 years. Thirty-three tumors involved the mandibular molar region andascending ramus (61.1%). The time to recurrence was from two months to 26 years with an average of 6.7
years. Several patients even had four times of recurrence. Twenty-five cases with odontogenic keratocyst(OKC) and seven cases of normal oral mucosa were used as control.
All

the

specimens

were

fixed

in

10%

formaldehyde

androutinelyparaffin-embedded

3-ammopropyl-triaceoxysilicone (APES) was smeared for slass retention. The sections were 4 µm thick.
Thirty sections were obtained in each case and three were randomly selected for experiment. The sections
were spread with double distilled water containing 0.1% diethyl oxydiformate (DEPC). All the specimens
were subjected to detection of hTR, hTERT and p53 protein, although the cases were not exactly the same
due to staining problems.
Main Reagents
MAb of p53, S-P supersensitivity assay and DAB ingrain agent were products from Maxim, and p53 mRNAprobes were purchased from the Department of Pathology, Medical College, Peking University. The plasmid
containing hTR gene (pGR) was a present from Dr. Geron. The bacterium was conventionally transformed andthe plasmid amplified. The restricted incision enzyme Mlu I was used for linearization of the plasmid, then thehTR cRNA probes were prepared utilizing ex vivo transcription. According to the hTERT mRNA sequencereported by Nakamura et al., a pair of primer was designed both in the up and down stream of the open reading
frame with a length of 626 bp. To facilitate the subsequent cloning of the PCR products, Sal I and Bamh I
enzyme cutting situs were imported at 5’ end of the two primers with preservation of four additional basic
groups, to warrantee the effective cutting by the restricted incision enzymes. The primers were synthesized by
Shanghai Sai Bai Sheng (SBS) Company with the sequence as follows: 5’-TATAGTCGACGTGGAAGATGAGCGTGCGG3’;3’-primer

5’ACACGGATCCACCTTGACAAAGTACAGCTC3’.

Orentational cloning was adopted with Sal I and Bamh I cutting the PCR products and pBLuescript SK+vector.
The sub products were retrieved and purified, processing according to the proposed steps of T4 ligase by
Gibco-BRL Co., coupled reaction at 26˚C to 28˚C for two hours, then the coupled mixture was used fortransforming Escherichia coli JM109 strain, with the latter spreading on the agar plate containing Ampicillin,
cultured at 37˚C overnight. Individual colony was chosen for further culture, and the plasmid DNA wasprepared with basic fragmentation. Sal I and Bamh I were used for cutting and probation. The positive clone
was filtrated, and the plasmid was linearized with incision enzymes. Ex vivo transcription was used to prepare
biotin hTERT cDNA probes.

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Int Chin J Dent 2004; 4: 27-33.

Immunohistochemical Assay
According to the specifications of the reagent made by Fuzhou Maixin company, Fujian, P. R. China,cases of breast cancer were used as positive control, while PBS substituted the first antibody as negative
control.
In Situ Hybridization
The paraffin-embedded blocks were routinely sectioned and dewaxed. After hydration with alcohol, thesections were treated with chlorhydric acid (0.1mol/L), digested with protease K (1:100) at 37˚C for 15
minutes, then fixed with 4% paraformaldehyde at -20˚C for 10 minutes, dehydrated with 90% icy alcoholfor one to two minutes, placed in wet boxes containing 50% formamide, dropwising 20 µL hybridism
liquid (containing 50% formamide, ssDNA 2 µL, probe 2 µL, 50 mmol/L DTT, 5xDenharts, 5% dextron
sulfate, 4xSSC) in each section. The sections were sealed with sealing films, and the wet boxes wereplaced in the warm closets at 37˚C for 20 hours. After hybridization, the sections were rinsed with 50%
formamide, 2xSSC containing 50% formamide at 37˚C for 30 minutex1, rinsed with 2xSSC at 42˚C for 15
minutesx3, 0.1xSSC at 37˚C for 15 minutesx1, Buffer I (100 mmol/L Tris-cl pH>0.5, 150 mmol/L NaCl)
for 5 sx1. Sera equinum (1:100) was added, sealed at room temperature for 40 minutes. Anti-Dig-AP(1:500) was added and sealed at room temperature for another 60 minutes, stained with NBT/BCIP for 30
minutes, sealed with gelatin. Oral squamous cell carcinoma was served as positive control; sections
without addition of probes were as negative control.
Outcome Evaluation
Positive p53 protein was brown-yellow particles in the nucleus.

Each section was observed anddetermined under low power lens (40x, 100x), then the positive nuclei were counted under high power lens
(200x). Double blind was adopted in counting and the average values were calculated from three fields ofvision. Positive hTR and hTERT was considered when purple-blue particles present in the cytoplasm.
Less than 10% was (-), 10% to 40% was (+), 40% to 70% was (++), and more than70% was (+++).
Statistical analysis
SPSS 10.0 software was used for chi square test and Kendall Pearson correlation analysis. P<0.05 wasconsidered to be significantly different.

Results
In the normal oral mucosa, hTR and hTERT were weakly positive in the basal layer of the epithelium in
2/7 and 1/3 specimens, respectively. The p53 protein was lightly positive in the nucleus of the basal cells
in one section. The expression rate of hTR and hTERT in OKC was 81.3% (13/16) and 87.5% (14/16),
respectively, from the basal layer to the accessory basal layer, mainly weakly positive and intermediately
positive. The expression of hTR and hTERT in keratinocytes was negative (Fig. 1). The p53 protein was
negatively stained in 14 OKCs, while in 11 OKCs, the nucleuses of the basal cells were positively stained(Fig. 2) but weak. HTR and hTERT was positively expressed in 51 of 54 Abs (94.4%), dominated byintermediate and strong staining. The stain was mostly distributed in the peripheral column cells or cube

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Int Chin J Dent 2004; 4: 27-33.

cells as well as the scattered or densely proliferated polygon cells and spindle cells. The keratinizeddegeneration-shape cells and granular cells were not stained (Fig. 3). Most of the matrix had no signals of
hTR and hTERT mRNA, except some inflammatory cells. In areas of fibrocyte densely proliferating or
active, signals of hTR and hTERT were noted in two and six cases, respectively. The p53 positive protein
was mainly distributed in the nucleuses of the peripheral and stellate cells of ameloblastoma, in particular,
in the zones with extensive cell proliferation. Similarly, the keratinized degeneration-shape cells andgranular cells were seldom or not stained (Fig. 4). In contrast, p53 mRNA was positive in the cytoplasm of
granular cells, weakly positive in most ABs and intermediately positive in fewer ABs. In 13/49 (26.5%)
cases, p53 protein was negative while hTR and hTERT was positive.

1
2
Fig. 1. The hTR mRNA was strongly positive in basal cells and negative in keratinocyte in OKC(in situ hybridization, x400).
Fig. 2. The p53 protein was positive in cell nucleus of OKC (S-P method, x160).

3
4
Fig. 3. The hTERT mRNA was strongly positive in peripheral cells and negative in keratinizeddegeneration-shape cells of AB (in situ hybridization, x160).
Fig. 4. The p53 protein was positive in peripheral and proliferative stellate central cells, negative
or lightly positive in keratinized degeneration-shape cells of AB (S-P method, x200).
Clinocobiologically, in 31 cases with primary AB, the positive rate of hTR and hTERT was 96.7%(30/31) and 93.5% (29/31), respectively; while in 19 cases with recurrent AB, the positive rate of hTR andhTERT was 89.4% (17/19) and 94.9% (18/19), respectively. In four cases with malignant AB, the positive
rate was 100% (4/4). The positive rate and intensity of hTERT mRNA raised with recurrence of AB and
the increased grades of malignancy, while there was no significant relation in hTR between primary cases30Zhong et al.

Int Chin J Dent 2004; 4: 27-33.

and recurrent cases, except significant differences between primary cases and malignant cases, recurrent
cases and malignant cases (p<0.05), which was detailed in Table 1. Similarly, in cases with recurrent andmalignant ABs, p53 protein was significantly increased, compared with primary ABs, (p<0.01, Table 2).
Table 1.

Relationship between the expression of hTR and hTERT mRNA, and clinical biologicalbehavior of AB.
hTR mRNAhTERT mRNA
Groups
No.
+
++
+++
+
++
+++
Primary
31
1
2
19
9
2
6
13
10
Recurrent 19
2
0
3
14
1
2
2
14
Malignant 4
0
0
1
3
0
0
0
4
2

2

2

Note: hTR x =14.419, p<0.025; x =12.921, p=0.005 between primary and recurrent AB. hTERT x =8.687, p<0.05 between
primary and recurrent AB.

Table 2. The expression of p53 protein with clinical grades of AB.
GroupsNo.
X (SD)
Primary22
13.7 (5.6)
Recurrent
24
52.2 (20.1)
Malignant3
368.3 (31.0)

P value
F=95.717
p<0.001

Note: p<0.01 between primary AB and recurrent AB, primary AB and malignant AB, recurrent AB and malignant AB.

Table 3.
hTR
+
++
+++

The correlation of the expression of hTR and hTERT in AB.
hTERT
+
++
7
3
1
0
3
2
3
4
11
1
2
9

+++
0
1
10
20

Note: Kendall correlation analysis, rs=1.000, p=0.001.

Table 4.

The expression of hTR, hTERT mRNA and p53 protein in AB, OKC and normal oral
mucosa.
No. hTR mRNA
hTERT mRNA
P53 (x200)
X
+
++
+++
+
++
+++
Oral mucosa
771.4 0
28.6
0
85.7
14.3
0
0
1.4000OKC
1618.8 25
18.8
37.5
12.5
18.8
50
18.8
2.0800Primary AB
31
3.2
6.5
61.3
29.0
31
6.5
19.4
41.9
13.7136
Recurrent AB
1910.5 0
15.8
73.7
5.3
10.5
10.5
73.7
52.2083
Malignant AB 4
0
0
25.0
75.0
0
0
0
100
368.3333
In addition, the expression of hTR, hTERT mRNA and p53 protein in AB was not correlated to theprimary sites, X-ray features (monolocular or multilocular) and histopathologic patterns (follicular,
plexiform, granule cell subtype and desmoplastic) (p>0.05).
Clinicopathologically, several follicular tumors underwent granular cell transformation, but only
accounting 1/3 of the lesions, therefore, it cannot be regarded as an independent entity. The cases withdesmoplastic pattern were too small; no significant correlation was noted statistically. The expression andcorrelation of hTR and hTERT in AB was shown in Table 3. It was noted from Table 3 that the expressionof hTR and hTERT in AB was positively correlated. Also, the expression of hTR, hTERT and p53 hadpositive correlation (p<0.001, Table 4).

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Int Chin J Dent 2004; 4: 27-33.

By Pearson correlation analysis, in hTR, rs=0.536, the strong positive rate of hTR (+++) was positively
correlated with the expression of p53 (p<0.001), while in hTERT, rs=0.754, the strong positive rate ofhTERT (+++) was positively correlated with the expression of p53 (p<0.001).

Discussion
The p53 gene is the most correlative gene with human neoplasms discovered so far. It is generally
believed that p53 belongs to tumor suppressor gene. Only when p53 undergoes mutation or interrelation
with other proteins, can p53 lose its normal function and promote tumor generation.2 The wild type p53
exerts negative regulation on cell proliferation.

It was demonstrated by massive investigations that

telomerase is one of the most specific and sensitive molecular markers for human tumors until now,3,4 andthe activity of telomerase can be used as a marker of malignant tumor and predication of prognosis. The
regulation of mechanism of telomerase remains unclear.
5

protooncogene C-myc activated telomerase.

It was reported that the overexpression ofAntisensedigonucleotides against C-myc can down regulate

the activity of telomerase in leukemia cells.6 Kumamoto et al.7 showed that C-myc and hTERT had asimilar expression in AB. Other study reported that PMA, the agonist of protein kinase C (PKC) can
improve the activity of telomerase,8 bcl-2 and Rb gene were also involved in the event. The expressions of
p53 and telomerase were different in various reports, some believed the expressions of p53, hTR andhTERT in malignant tumors were correlated,9,10 but some found no correlation with p53.11 The expressionof p53, hTR and hTERT in AB and OKC has not been reported previously.
Our study showed that the positive situs of p53, hTR and hTERT were similar in the peripheral andstellate layer of AB, while the keratinized degeneration-shape cells and granular cells were seldom or not
stained. This implied that in the same tumor, different cells had different activity and differentiation.
Therefore, the expression of p53, hTR and hTERT displayed heterogenicity. The present study confirmedthat keratinized degeneration-shape cells and granular cells represent terminal differentiation of cells.12
Similarly, the activated cells of hTR and hTERT in OKC were mainly situated in the basal layer, other thanin the corneal layer, which was in consistence with p53 protein mostly expressing in the basal layer,
reflecting a quite high proliferation of cells in the basal layer of OKC, according with the expression of
bcl-2.13 Compared with OKC and normal oral mucosa, the expression of p53 in AB was highest (85.7%,
42/49), the positive rate of hTR and hTERT was as high as 94.4% (51/54), while in malignant AB, hTR andhTERT was 100% strongly positive, and the cells with positive staining of p53 protein increased as well,reaching 300 to 400 in each high power field, which enunciated that the high expression of p53 was inconsistence with the strongly positive staining of hTR and hTERT (p<0.001). El-Sissy14 proposed that theoverexpression of p53 reflected its important role in tumor growth and carcinogenesis. In one study,15 thepositive staining of p53 was shown significantly stronger in AB than in amelificator and dentate laminae.
Invasion is one of the principal biologic characteristics of malignant tumors. Although regarded as benign
tumor by WHO, AB has unique biologic behavior, being borderline tumor in fact. Clinically, AB grows
invasively like malignant tumors, with a high recurrence rate after intervention. The high expressions of32

Zhong et al.

Int Chin J Dent 2004; 4: 27-33.

hTR and hTERT mRNA as well as p53 protein in AB demonstrated in this study provided evidence for its
invasive clinical behaviors.
It was demonstrated that the expression of hTR and hTERT was not consistent with the activity oftelomerase,16 however, the expression of hTR and hTERT in AB and OKC was coherent, and related to thehigh expression of p53 protein. In few cases, the expression of p53 was negative, yet the expression ofhTR and hTERT was positive, which indicated that the activity of telomerase was still regulated by otherintracellular signal transductions and engaged in carcinogenesis coordinately.

References
1. Kim NW, Piatyszek MA, Prowse KR, et al. Specific association of human telomerase activity with immortal cells andcancer. Science 1994; 266: 2011-5.
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3. Mao L, El-Naggar AK, Fan YH, et al. Telomerase activity in head and neck squamous cell carcinoma and adjacenttissues. Cancer Res 1996; 56: 5600-4.
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Reprint request to:
Dr. Ming Zhong
Department of Pathology, College of Stomatology, China Medical University
117 Nan Jing North Street, Heping District, Shenyang, 110002, P. R. China
Fax:+86-24-23410142 E-mail: zhongming1234@163.net
Received January 17, 2004. Revised February 24, 2004. Accepted March 1, 2004.
Copyright ©2004 by the Editorial Council of the International Chinese Journal of Dentistry.

33