Journal of Dentistry Defence Section

ORIGINAL ARTICLE
Year
: 2022  |  Volume : 16  |  Issue : 1  |  Page : 12--18

Analysis of cell cycle activity by expression of p-53 protein in different gingival overgrowths: An immunohistochemistry-based pilot study


Sunny Bhatia, Manish Mukherjee, Partha Roy, Parvez Shaikh, Priyanka Prakash 
 Department of Periodontology, Army Dental Centre, Research and Referral, New Delhi, India

Correspondence Address:
Sunny Bhatia
Department of Periodontology, Army Dental Centre, Research and Referral, New Delhi
India

Abstract

Context: It is a crucial protein expressed in cells to prevent tumor formation. Hence, the study of expression of p-53 protein is of high importance in gingival cells in various conditions such as drug-induced gingival enlargement and nondrug-induced gingival enlargements. Aims: This study aimed to analyze the cell cycle activity for the p-53 protein expression in the cells of drug-induced and nondrug-induced gingival overgrowths as compared to healthy subjects. Settings and Design: This was a single-blinded observational pilot study. Subjects and Methods: Patients were included on the basis of various inclusion and exclusion criteria and divided into three groups. Inclusion Criteria:
  1. Group A (patients with drug-induced gingival overgrowth)
    • Patients on drugs such as anticonvulsant, immunosuppressants, and calcium channel blockers ≥6 months.
  2. 2. Group B (patients with nondrug induced gingival overgrowth)
    • Patients not on drugs such as anticonvulsants, immunosuppressants, and calcium channel blockers ≥1 year.
  3. Group C (healthy subjects)
Exclusion Criteria: Patients on antibiotics and edentulous patients. Protocol:
  • Gingival samples were collected after obtaining consent from patients and were stored in 10% formalin
  • The samples were blinded and the assay for the expression of p-53 was done by immunohistochemistry along with histopathological examination.
Statistical Analysis Used: Statistical analysis was conducted by using SYSTAT-13 software. Results: Expression of p-53 protein in each group was analyzed after preparing slides for immunohistochemistry examination. A total of 10 samples were analyzed in each group to check for the expression of p-53 protein in basal as well as suprabasal cell layers. Each sample was then correlated with the corresponding slides for histopathological examination of each sample for each group. Conclusions: The p-53 protein expression in the hyperplastic gingival epithelia noted in our present study suggests that gingival hyperplasia induced by drugs or by inflammation has possible implications for pathogenesis accompanied with impaired DNA. On the other hand, our study also suggests that the growth arrest takes place in the representative rete pegs deeply elongated into lamina propria of hyperplastic gingival tissues, which contributes to the inhibition of DNA damaged cell expansion within gingival tissues followed by promoting the tumorigenic aberrations of gingival hyperplasia.



How to cite this article:
Bhatia S, Mukherjee M, Roy P, Shaikh P, Prakash P. Analysis of cell cycle activity by expression of p-53 protein in different gingival overgrowths: An immunohistochemistry-based pilot study.J Dent Def Sect. 2022;16:12-18


How to cite this URL:
Bhatia S, Mukherjee M, Roy P, Shaikh P, Prakash P. Analysis of cell cycle activity by expression of p-53 protein in different gingival overgrowths: An immunohistochemistry-based pilot study. J Dent Def Sect. [serial online] 2022 [cited 2022 Jun 30 ];16:12-18
Available from: http://www.journaldds.org/text.asp?2022/16/1/12/342641


Full Text



 Introduction



p-53 protein is encoded by the p-53 tumor suppressor gene which is located on the short arm of chromosome 17. The amplification of p-53 gene arises and the expression of wild-type p-53 protein is enhanced when DNA strands are impaired by DNA-damaging agents such as ultraviolet (UV) rays and chemical carcinogens.

The increased wild-type p-53 protein arrests cells in G1 phase of the cell cycle and allows time to repair the damaged DNA prior to entering into S phase. If the mutations of genes are so severe that it is impossible to repair the DNA damage, the increased wild-type p-53 protein induces apoptosis of transformed cells. Therefore, p-53 gene prevents the expansion of cells which possess the mutated genes in the genome and is referred to as “guardian of the genome.”

Two possibilities have been proposed for immunodetection of p-53 protein expression. One is that wild-type p-53 protein is overexpressed just after DNA damage. The other is that the half-life time of mutant p-53 protein is elongated and it remains within the nuclei. Therefore, the expression of p-53 protein can be one of the markers to examine the state of impaired DNA.

Hence in this study, patients with different etiology of gingival overgrowth were selected and expression of p-53 was checked in the gingival samples by doing a immunohistochemistry analysis.

 Subjects and Methods



Gingival specimens

This retrospective case–control study was conducted by selecting 30 patients from the central OPD of ADC R&R. These patients were divided into three groups with 10 patients in each group, as depicted in [Figure 1].{Figure 1}

These patients were included in the groups based on the following criteria:

  1. Group A (patients with drug-induced gingival overgrowth)
    • ≥25 and ≤55 years of age
    • Patients on drugs such as anticonvulsant, immunosuppressants, and calcium channel blockers ≥6 months
  2. B (Patients with nondrug-induced gingival overgrowth)
    • ≥25 and ≤55 years of age
    • Patients not on drugs such as anticonvulsant, immunosuppressant, and calcium channel blockers ≥1 year
    • Inflammatory type of enlargement
    • Gingival carcinomas.
  3. Group C (healthy subjects)
    • Gingival samples were collected during crown lengthening procedure
    • Gingival samples on the extracted teeth in orthodontics patients.


The patients were excluded based on the following criteria:

  • Pregnant and lactating women
  • Patients having teeth with endo-perio lesions
  • Patients using tobacco or tobacco-related products
  • Patients having bleeding disorders
  • Patients requiring antibiotic prophylaxis prior to any invasive dental care.


Methodology

Tissue samples of gingival overgrowth were taken from patients categorized into three groups. Each tissue sample was split into two samples by No. 15 BP blade. The two samples obtained were immersed in two sample bottles with 10% formalin each [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]. Samples were then sent to the laboratory for immunohistochemistry and histopathological examination.{Figure 2}{Figure 3}{Figure 4}{Figure 6}{Figure 6}

The gingival tissues were then fixed in wax blocks and sectioned using microtomes and mounting on the charged slide. The charged slides were then taken up for chromogenic staining for the expression of p-53 protein and then examined under microscope [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12].{Figure 7}{Figure 8}{Figure 9}{Figure 10}{Figure 11}{Figure 12}

 Results



Expression of p-53 protein in each group was analyzed after preparing slides for immunohistochemistry examination. A total of 10 samples were analyzed in each group to check for the expression of p-53 protein in basal as well as suprabasal cell layers. Each sample was then correlated with the corresponding slides for histopathological examination of each sample for each group.

Group A: Drug-induced gingival overgrowth

After histopathological and immunohistochemistry examination, it was found that P-53 was expressed in basal as well as suprabasal layers in all samples [Figure 13] and [Figure 14].{Figure 13}{Figure 14}

Group B: Nondrug-induced gingival overgrowth

After histopathological and immunohistochemistry examination, it was found that P-53 was expressed in basal as well as suprabasal layers in all samples [Figure 15] and [Figure 16].{Figure 15}{Figure 16}

Group C: Healthy subjects

After histopathological and immunohistochemistry examination, it was found that P-53 was expressed in basal as well as suprabasal layers in all samples [Figure 17] and [Figure 18].{Figure 17}{Figure 18}

Statistical analysis

Statistical analyses were done and a comparison was made to analyze the expression of p-53 in each group and also a intergroup comparison was carried out using SYSTAT-13® software. The expression of p-53 in each group was tabulated [Table 1] and [Table 2] and graphs were plotted. The statistical analysis of intergroup comparison was done using Freidman two-way analysis of variance.{Table 1}{Table 2}

In Group A and Group B, the expression of p-53 was found to be expressed in basal and suprabasal layers. A more strong expression was seen in case of samples taken in Group A. The samples taken from Group C showed a mild expression in basal cell layer only in three samples and the rest of the samples were negative for the expression of p-53 protein. The same can be seen on plotting a graph using Freidman two-way analysis of variance [Graph 1].[INLINE:1]

 Discussion



The expression of p-53 protein and mutations of the p-53 gene have been confirmed as critical processes in tumorigenesis.[1],[2] Intense p-53 protein expression has been observed in oral premalignant lesions and carcinomas[3],[4] as well as in nonoral carcinomas.[4],[5],[6] Mutations of the p-53 gene have also been associated with oral carcinomas.[7]

In addition to neoplastic tissues, scattered p-53 protein expression has been noticed in the histologically normal epithelia adjacent to oral carcinomas[8],[9],[10] and other carcinomas.[11] The nonneoplastic epithelia adjacent to basal cell carcinomas and laryngeal carcinoma[12] and pharyngeal carcinoma[13] have also been observed to reveal mutations of the p-53 gene.

Moreover, it has been reported that a normal epidermis, when exposed to UV radiation, gives rise to DNA damage and shows sporadic patterns of p-53 protein expression and mutations of the p-53 gene.[14],[15],[16]

In the present study, the immunolocalization of p-53 protein in the epithelia of hyperplastic gingival tissues was also found sporadically. Thus, the hyperplastic epithelia may, in part, undergo DNA damage by genotoxic stress of the drugs causative of gingival hyperplasia especially in case of Group A where a strong expression of p-53 protein was found in the basal and suprabasal cell layer of the gingival epithelium.

Saito et al.[17] have ascertained the similar expression of p-53 and ki57 antigens in the gingival cells.

Group B also depicted the presence of p-53 but was weakly expressed when compared to Group A suggestive of some kind of DNA damage or the role of expression of p-53 in the inflammatory process. A few samples from Group C which included healthy gingival tissues also expressed very weakly the presence of p-53 represent a subclinical inflammation. The absence of expression of p-53 was also seen in most of the samples of Group C.

The histological features of proliferating fibroblasts in the lamina propria of hyperplastic gingival tissues were comparable and different from the healthy tissues where the absence of hyperplasia of the gingival epithelium was seen along with elongated rete pegs extending into the lamina propria.

 Conclusions



The p-53 protein expression in the hyperplastic gingival epithelia noted in our present study suggests that gingival hyperplasia induced by drugs or by inflammation has possible implications for pathogenesis accompanied with impaired DNA. On the other hand, our study also suggests that the growth arrest takes place in the representative rete pegs deeply elongated into lamina propria of hyperplastic gingival tissues, which contributes to the inhibition of DNA damaged cell expansion within gingival tissues followed by promoting the tumorigenic aberrations of gingival hyperplasia. However, further studies on a larger population will be required to understand the entire role of control and expression of p-53 protein.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1Gottlieb TM, Oren M. p53 in growth control and neoplasia. Biochim Biophys Acta 1996;1287:77-102.
2Kaur J, Srivastava A, Ralhan R. Overexpression of p53 protein in betel- and tobacco-related human oral dysplasia and squamous-cell carcinoma in India. Int J Cancer 1994;58:340-5.
3Kuttan NA, Rosin MP, Ambika K, Priddy RW, Bhakthan NM, Zhang L. High prevalence of expression of p53 oncoprotein in oral carcinomas from India associated with betel and tobacco chewing. Eur J Cancer B Oral Oncol 1995;31B: 169-73.
4Charpin C, DeVictor B, Andrac L, Amabile J, Bergeret D, LaVaut MN, et al. p53 quantitative immunocytochemical analysis in breast carcinomas. Hum Pathol 1995;26:159-66.
5Müller W, Borchard F. Prognostic influence of p53 expression in gastric cancer. J Pathol 1996;178:255-8.
6El-Naggar AK, Lai S, Luna MA, Zhou XD, Weber RS, Goepfert H, et al. Sequential p53 mutation analysis of pre-invasive and invasive head and neck squamous carcinoma. Int J Cancer 1995;64:196-201.
7Zariwala M, Schmid S, Pfaltz M, Ohgaki H, Kleihues P, Schäfer R. p53 gene mutations in oropharyngeal carcinomas: A comparison of solitary and multiple primary tumours and lymph-node metastases. Int J Cancer 1994;56:807-11.
8Shin DM, Kim J, Ro JY, Hittelman J, Roth JA, Hong WK, et al. Activation of p53 gene expression in premalignant lesions during head and neck tumorigenesis. Cancer Res 1994;54:321-6.
9Nakanishi Y, Noguchi M, Matsuno Y, Saikawa M, Mukai K, Shimosato Y, et al. p53 expression in multicentric squamous cell carcinoma and surrounding squamous epithelium of the upper aerodigestive tract. Immunohistochemical analysis of 95 lesions. Cancer 1995;75:1657-62.
10Piffkó J, Bankfalvl A, Öner D, Berens A, Tkotz T, Joos U, et al. Expression of p53 protein in oral squamous cell carcinomas and adjacent non-tumorous mucosa of the mouse: An archival immunohistochemical study using wet autoclave pretreatment for antigen retrieval. J Oral Pathol Med 1995;24:337-42.
11Urano Y, Asano T, Yoshimoto K, Iwahana H, Kubo Y, Kato S, et al. Frequent p53 accumulation in the chronically sun-exposed epidermis and clonal expansion of p53 mutant cells in the epidermis adjacent to basal cell carcinoma. J Invest Dermatol 1995;104:928-32.
12Pruneri G, Pignataro L, Fracchiolla NS, Ferrero S, Capaccio P, Carboni N, et al. p53 protein expression in laryngeal squamous cell carcinomas bearing wild-type and mutated p53 gene. Histopathology 1996;28:513-9.
13Nees M, Homann N, Discher H, Andl T, Enders C, Herold-Mende C, et al. Expression of mutated p53 occurs in tumor-distant epithelia of head and neck cancer patients: A possible molecular basis for the development of multiple tumors. Cancer Res 1993;53:4189-96.
14Campbell C, Quinn AG, Angus B, Farr PM, Rees JL. Wavelength specific patterns of p53 induction in human skin following exposure to UV radiation. Cancer Res 1993;53:2697-9.
15Hall PA, McKee PH, Menage HD, Dover R, Lane DP. High levels of p53 protein in UV-irradiated normal human skin. Oncogene 1993;8:203-7.
16Berg RJ, van Kranen HJ, Rebel HG, de Vries A, van Vloten WA, Van Kreijl CF, et al. Early p53 alterations in mouse skin carcinogenesis by UVB radiation: Immunohistochemical detection of mutant p53 protein in clusters of preneoplastic epidermal cells. Proc Natl Acad Sci U S A 1996;93:274-8.
17Saito K, Mori S, Tanda N, Sakamoto S. Expression of p53 Protein and Ki-67 Antigen in Gingival Hyperplasia Induced by Nifedipine and Phenytoin. J Periodontol 1999;70:581-6.