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ORIGINAL ARTICLE
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Immunohistochemical expression of p53 and murine double minute 2 protein in odontogenic keratocyst versus variants of ameloblastoma


 Department of Oral Pathology and Microbiology, ITS Dental College, Muradnagar, Ghaziabad, Uttar Pradesh, India

Date of Submission09-Dec-2018
Date of Decision17-Apr-2019
Date of Acceptance11-May-2019
Date of Web Publication29-Jan-2020

Correspondence Address:
Anshi Jain,
Department of Oral Pathology and Microbiology, ITS Dental College, Muradnagar, Ghaziabad, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_659_18

 > Abstract 


Introduction: Oncogenes and tumor suppressor genes play a major role in cancer formation, growth, and progression. One of the important findings in this area is that murine double minute 2 (MDM2) oncogene is a negative regulator of wild-type p53. In tumors, expressing wild-type p53, inhibition of MDM2 expression will stabilize p53 and allow it to perform its proapoptotic function, while simultaneously preventing MDM2 from exerting its p53-independent oncogenic effects. The intracellular levels of p53 are tightly regulated by MDM2, as it is a key player in autoregulatory feedback loop under nonstressed conditions. The p53-MDM2 relationship is vital not only for essential functions of the cell, but it also appears to be an integrated part of the complex cellular network which supports the importance of this affair and is a hallmark for its coexistence.
Subjects and Methods: This study was designed to identify immunohistochemically the expression of p53 and MDM2 gene using monoclonal antibody in 60 cases of formalin-fixed paraffin-embedded tissue blocks, of which 20 cases were of solid multicystic ameloblastoma (SMA), 20 cases were of odontogenic keratocyst (OKC), and 20 cases were of unicystic ameloblastoma (UA).
Results: Immunoexpression of p53 and MDM2 was highest in OKC followed by SMA and was minimum in UA. Further results showed positive correlation between both the molecules.
Conclusion: The studied showed that the relationship has a significant role in cancer etiology and progression and therefore is an important topic for future research which should help in the development of new therapeutic agent against cancer.

Keywords: Murine double minute 2, odontogenic keratocyst, p53, solid multicystic ameloblastoma, unicystic ameloblastoma



How to cite this URL:
Singh A, Jain A, Shetty DC, Rathore AS, Juneja S. Immunohistochemical expression of p53 and murine double minute 2 protein in odontogenic keratocyst versus variants of ameloblastoma. J Can Res Ther [Epub ahead of print] [cited 2020 Feb 29]. Available from: http://www.cancerjournal.net/preprintarticle.asp?id=277278




 > Introduction Top


The DNA within our cells is reliably being mapped and deciphered. This work combined with new results from molecular and cellular biology is enabling researchers to reconstruct the inner workings of cells in unprecedented detail. Scientists worldwide are beginning to build a holistic framework for understanding the human organism, one that integrates the distinct yet interrelated roles of DNA genes, the cell, the body, and the environment. With it comes a better understanding of the cellular origins of many diseases including the origins of cancer. The cellular programs of proliferation, differentiation, senescence, and apoptosis are intimately linked to cell-cycle regulatory machinery, and the dysregulation of this cell-cycle machinery is a fundamental hallmark of cancer progression.[1]

Three classes of normal regulatory genes, “the growth promoting proto-oncogenes, the growth inhibiting cancer suppressor genes (antioncogenes), and genes that regulate programmed cell death or apoptosis,” are principal targets of genetic damage. Activation of proto-oncogenes that promote cell growth in combination with the inactivation of tumor suppressor genes that inhibits cell growth and induction of programmed cell death or apoptosis leads to tumor progression and malignancy.[2]

Perhaps, the most important in this regard is abnormalities of p53 tumor suppressor gene, which acts as a molecular policeman “monitoring the integrity of the genome.” They are the principal mediator of growth arrest, senescence, and apoptosis in response to a broad array of cellular damage.[2] Depending on the conditions of cell growth, the type and duration of stress or DNA damage p53 selectively activate a different subset of target genes which can cause either apoptosis, growth arrest, altered DNA repair, or altered differentiation.[2]

There is increasing evidence that the tumor suppressor function of p53 can be inhibited in the absence of mutation and conceivably because of the murine double minute 2 (MDM2) oncogene which is a negative regulator of p53.[2]

Since its first discovery 18 years ago, MDM2 oncogene has been a research subject in cancer biology. The most exciting finding is the MDM2-p53 autoregulatory feedback loop. This paradigm represents the best-studied relationship between a tumor suppressor gene and an oncogene which functions primarily as an E3 protein ligase where MDM2 gene is a target for direct transcriptional activation by p53 and MDM2 protein in turn negatively regulates p53 by inhibiting p53 functions and inducing p53 degradation intracellularly.[3]

The intimate relationship between these two partners has expanded to include almost every cellular biological system, from development to growth control and programmed cell death. The affair between MDM2 and p53 is closely controlled by a complex array of posttranslational modifications which, in turn, dictates the stability and activity of p53 and MDM2.[2]

Overexpression of MDM2 has been demonstrated in a variety of human tumors, and it is important to note that in many cases, MDM2 is upregulated by mechanism other than gene amplification including increased transcription and enhanced translation.[4] Several researchers have shown that overexpression of MDM2 is associated with poor prognosis.[5] The odontogenic keratocyst (OKC) first described by Philipsen in 1956 and is defined as “a benign uni or multicystic, intraosseous tumor of odontogenic origin with a characteristic lining of parakeratinized stratified squamous epithelium and potential for aggressive, infiltrative behavior [Figure 1]a.
Figure 1: (a) H and E-stained section of odontogenic keratocyst. (b) Positive nuclear immunoexpression of murine double minute 2 in basal and parabasal layers in odontogenic keratocyst (×40). (c) Positive p53 immunoexpression in basal and parabasal layers in odontogenic keratocyst (×40)

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Odontogenic tumors (OTs) being a topic of considerable interest in the research field. The term “tumor” used for OTs constitutes a group of heterogeneous lesions that range from hamartomatous or nonneoplastic tissue proliferations to malignant neoplasms with metastatic capabilities. Due to their specific structure and location, they have been identified and classified by pathologists into a separate group differing in histogenesis, biology, clinical manifestations, and radiological signs from other tumors developing in the oral cavity and facial bones.[6]

Ameloblastomas which constitute almost half (48.9%) of the OTs are a representative benign tumor of odontogenic epithelium.[4] Ameloblastoma is a benign, but locally invasive neoplasm derived from odontogenic epithelium is the most frequently encountered OT and has a high tendency to recur. It is a unique tumor in the oral and maxillofacial region exhibiting a wide range of different histological patterns with various proliferative activities in each type [Figure 2]a and [Figure 3]a.[6]
Figure 2: (a) H and E-stained section of unicystic ameloblastoma. (b) Positive nuclear immunoexpression of murine double minute 2 in the basal and parabasal layers in unicystic ameloblastoma (×40). (c) Positive p53 immunoexpression in the basal and parabasal layers in unicystic ameloblastoma (×40)

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Figure 3: (a) H and E-stained section of solid multicystic ameloblastoma. (b) Positive nuclear immunoexpression of murine double minute 2 in the basal and parabasal layers in solid multicystic ameloblastoma (×40). (c) Positive p53 immunoexpression in the basal and parabasal layers in solid multicystic ameloblastoma (×40)

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OT may also be a result of activation of proto-oncogene and decrease of tumor suppressor gene which has not been explained in detail in the past.[7],[8]

Hence, the present study was designed to study qualitative and quantitative immunoepxpresion of MDM2 and p53 in OKC and variants of ameloblastomas and comparatively analyzed the immunoexpression of these proteins to postulate the pathogenetic model and aggressive nature of these lesions.


 > Subjects and Methods Top


A total of 60 cases were included in the study which comprised 20 cases each of OKC, solid multicystic ameloblastoma (SMA), and unicystic ameloblastoma (UA) and fixed in 10% neutral buffered formalin, processed and embedded in paraffin wax following standard protocol. About 4-μ thick sections were taken for hematoxylin and eosin staining and immunohistochemistry procedure. Based on the literature review and consultation with expert statistician, the sample size was estimated by the G-power analysis at 95% confidence interval, i.e., t-tests means: the difference between two independent means (two groups) and power of 80% in consultation with expert statistician and was found to be significant for outcome of the study.

Study settings

Tissue specimens were retrieved from the archives of confirmed cases of SMA, UA, and OKC. The obtained tissues were fixed in 10% neutral buffered formalin. From each case of formalin-fixed paraffin-embedded tissue blocks, 3–4-μ thick sections were obtained. One set of slides was stained by hematoxylin and eosin for reconfirmation of histological diagnosis, whereas the other two sets were stained for MDM2 and p53 using the standard immunohistochemical method.

Immunohistochemistry procedure

Immunohistochemical analysis for MDM2 and p53 was performed on 3 μm thin paraffin sections which were obtained using a rotary microtome. In brief, following dewaxing, washing, and rehydration of the slides through xylene and graded alcohol concentrations, tris-buffered saline and phosphate-buffered saline at pH 7.2–7.4 were used for antigen retrieval. Slides were subsequently treated with 3% hydrogen peroxide to block endogenous peroxidase. Following incubation with the primary antibodies, MDM2 (BioGenex) and p53 (BioGenex), the secondary conjugate antibody was applied and followed by chromogen DAB and counterstaining with Mayer's hematoxylin.

Positive control

For MDM2 – human breast carcinoma tissue.

For p53 – human oral squamous cell carcinoma.

The sections were stained following the same laboratory procedure as study cases. MDM2 stained crisp brown color in the nuclei of tumor area, whereas p53 stained selectively tumor cells in oral squamous cell carcinoma.

Negative control

Omitting the use of primary antibody and carrying out the successive steps of immunohistochemistry, as usual, will give the negatively stained slides.

Immounohistochemical analysis

All the immunostained slides were viewed under the light microscope. Layer-wise immunoscoring for percentage of cell positivity and intensity for MDM2 and p53 were done as mentioned below.

Quantitative analysis

Selection of field

Stained sections were scanned at ×40 to determine areas that were positively stained. Five random representative fields at ×400 were selected and 200 cells were counted per high-power field. By selecting five random fields which were not in continuum, a possible error in recounting the same cells was minimized.

Counting of cells

The images of five high-power fields (×400) were obtained with a digital camera (Olympus E-PL3) attached to the research microscope and were transferred to a computer system for analysis which was done using a grid. A total of 1000 cells were counted in five high-power fields. Immonoscoring of p53 and MDM2 (nuclear staining) was done by counting the number of immunopositive cells layer wise (basal and parabasal) per 1000 total tumor cells in OKC, SMAs and UAs, and labeling Index was evaluated as follows:



Qualitative analysis

Qualitatively nuclear immunoscoring of MDM2 and p53 was done. The tissue was observed under light microscope at ×100 and ×400 for the intensity of staining and scored as follows: 0 – negative staining, + – mild positive, ++ – moderate positive, and +++ – strong positive.

Interpretation of agreement of numerical scoring for nuclear expression of MDM2 and p53 was done using the following kappa analysis [Table 1].{Table 1}

All the slides were viewed in research microscope, and the number of positive cells and grade of intensity of immunopositive cells was compared with biological behavior of SMA, UA, and OKC to immunoexpression of MDM2 and p53.

Statistical analysis

The resulting data were analyzed using SPSS software version 19 (SPSS Inc., 233 South Wacker Drive, 11th Floor, Chicago, IL). Data have been expressed as mean and standard deviation. Differences between the different variables were analyzed using the ANOVA test, Paired t-test, and post hoc test followed by a Bonferroni test. Spearman's rho coefficient was also calculated. Pearson's Chi-square test was carried out to determine the level of correlation or association between the groups understudy. P < 0.05 was considered statistically significant.


 > Results Top


The data analysis of our study shows that all the three lesions (OKC, SMA, and UA) are more common in older adults as compared to younger age group and were predominately seen in males as compared to females. Mandible is the most commonly affected site in cases of our study.

The mean labeling index of MDM2 immunoexpression in the three study groups depicts that 90% cases of SMA were immunopositive for MDM2 and had a maximum mean labeling index (46.70 ± 16.918), followed by OKC (37.50 ± 20.377) and was least in UA (23.00 ± 19.431). These results were statistically significant (P ≤ 0.05).

Moreover, post hoc comparison of MDM2 labeling index immunoexpression in the three study groups shows that the mean difference in numbers of cell showing MDM2 expression in SMA and OKC was 9.200. Statistically, comparison of the mean difference score of SMA and OKC was nonsignificant (P > 0.05). Similarly, the mean difference in a number of cells between SMA and UA was 23.700 which was statistically significant (P ≤ 0.05). Finally, between OKC and UA, the mean difference was calculated as 14.500 which is nonsignificant (P > 0.05).

Quantitatively analyzing the number of immunopositive MDM2 cells in the basal and parabasal layers of the three study groups depicted that in the basal layer, the expression of MDM2 was maximum in SMA (30.20 ± 11.043) followed by UA (17.35 ± 14.709) and was least in OKC (10.95 ± 6.708), and in the parabasal layer, OKC (26.45 ± 14.122) was maximum followed by SMA (16.50 ± 7.452) and was least in UA (5.56 ± 4.826). The results of this were statistically significant (P ≤ 0.05) [Figure 1]b, [Figure 2]b and [Figure 3]b.

Further, the results of the study show that the mean labeling index of p53 immunopositive cells was maximum in OKC (40.00 ± 18.411) followed by SMA (27.70 ± 18.703) and was least in UA (13.30 ± 16.774). The results are statistically significant (P ≤ 0.05) [Table 2].{Table 2}

Moreover, post hoc comparison of p53 labeling index immunoexpression in the study groups show that that the mean difference in number of cells showing p53 expression in SMA and OKC was −12.300 which was statistically nonsignificant (P > 0.05). Similarly, the mean difference in number of cells between SMA and UA was 14.400, which was statistically significant (P ≤ 0.05). Finally, between OKC and UA, the mean difference was calculated as 26.700, indicating statistically high significance (P ≤ 0.05).

Further, quantitatively analyzing the number of immunopositive p53 cells in the basal and parabasal layers of three study groups depicted that the expression of p53 was maximum in the basal layer of SMA (18.15 ± 12.351) followed by OKC (17.85 ± 10.932) and was least in UA (9.90 ± 12.468), and in the parabasal layer, maximum immunopositive p53 cells were seen in OKC (22.20 ± 14.085) followed by SMA (9.50 ± 6.477) and was least in UA (3.40 ± 4.333). The results were statistically significant (P ≤ 0.05) [Table 2] and [Figure 1]c, [Figure 2]c and [Figure 3]c.

Interobserver qualitative analysis of p53 and MDM2 in the study groups was done using Cohen's kappa analysis, and the results of these observations (p53 −0.911 and MDM2 0.910) show that there was excellent agreement between the observers which was statistically highly significant (P ≤ 0.05).

Qualitative analysis of p53 immunostaining in the study groups shows that of total 20 cases of each group, 3 cases of OKC, 6 cases of SMA, and 12 cases of UA show negative staining. Strong staining intensity of p53 was observed in maximum cases of OKC (10 cases) followed by SMA (6 cases) and was minimum in cases of UA (only 1 case). Seven of 20 cases of SMA and 5 out of 20 cases of UA depicted moderate staining intensity. The results were statistically significant (P< 0.05).

Similarly, the qualitative analysis of MDM2 immunostaining in the study groups shows that out of total 20 cases of each study group, 4 cases of OKC, 2 cases of SMA, and 8 cases of UA show negative MDM2 immunostaining. Strong immunostaining of MDM2 was observed in maximum cases of SMA (10 cases) followed by OKC (6 cases) and was minimum in UA (4 cases). Maximum cases of OKC (8 cases) and UA (5 cases) showed moderate staining intensity. The results were statistically nonsignificant (P > 0.05).

Further, in the study, the correlation coefficient of p53 and MDM2 was calculated between the three study groups. Analysis showed positive p53 correlation with MDM2 in cases of SMA, OKC, and UA with correlation coefficient of 0.272, 0.596, and 0.467, respectively. These results indicate that p53 works synergistically with MDM2 (p53 immuoexpression increase, there is proportional increase in MDM2 expression) in the three study groups [Figure 4].
Figure 4: Correlation between p53 and murine double minute 2 in odontogenic keratocyst, solid multicystic ameloblastoma, and unicystic ameloblastoma

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 > Discussion Top


OTs are a group of heterogeneous lesions derived from the epithelial or ectomesenchymal tissues or both which are part of the tooth-forming apparatus. They range from hamartomatous or nonneoplastic tissue proliferations to malignant neoplasms with metastatic capacity. Due to their specific structure and location, they have been identified and classified by pathologists into a separate group, differing in histogenesis, biology, clinical manifestations, and radiological signs from other tumors developing in the oral cavity and facial bones.[9]

Although the etiology and pathogenesis of OTs remain unknown, recent studies have identified various molecular alterations responsible for their development and progression.[10] Among the potentially oncogenic elements of cell-cycle machinery, the expression of p53 and MDM2 in association with cell-cycle progression and apoptosis suggests that both play a central role in cancer development and progression.[5] An accumulating number of observations have implicated aberrant expression of p53 and MDM2 oncogene in pathogenesis of human neoplasms.[11]

The solid or multicystic ameloblastoma is a benign epithelial OT of the jaws. SMA may also arise as a result of neoplastic changes in the lining or wall of a nonneoplastic odontogenic cyst, in particular, dentigerous and OKCs.[2] Signaling pathway such as wingless-related integration site (WNT) and protein kinase B and growth factors such as fibroblast growth factor play a pivotal role in the pathogenesis of solid type of ameloblastoma.[12]

The aggressive behavior and high recurrence rate of the OKC suggest a true neoplastic potential and prompted the World Health Organization Working Group to classify the OKC as a benign tumor with odontogenic epithelium and mature, fibrous stroma without odontogenic ectomesenchyme.[13]

UA represents an ameloblastoma variant presenting as a cyst that shows clinical and radiologic characteristics of an odontogenic cyst. Three pathogenic mechanisms for the evolution of UA are from reduced enamel epithelium, from dentigerous cyst, and due to cystic degeneration of solid ameloblastoma.[12]

p53 is the most commonly mutated gene in human cancers and more than 50% of human cancers contain p53 mutation. The p53 tumor suppressor is present at a low concentration in normal cells. Stress, such as hypoxia or DNA damage, causes p53 to accumulate in the nucleus, where it is active.[14] Depending on the cellular stress and cell type, the activation of p53 can lead to various responses, for example, DNA damage might result in growth arrest to allow for repair of the damage, or apoptosis, both of these responses aim to prevent damaged cells from proliferating and passing mutations onto the next generation. p53 acts as a transcription factor and is able to activate many genes to induce these specific functions. As cells that lack functional p53 are unable to respond appropriately to stress, they can accumulate mutations that favor the development of cancer.[14]

The MDM2 gene as the name reflects was in fact localized on double-minute chromosomes (DMs).[15] It was originally cloned as an amplified gene in the tumorigenic 3T3 DM murine cell line localized on chromosome 12q 13–15.[12] This chromosomal position has been shown to be altered in many human cancers.[15] The MDM2 oncogene is a determinant of embryogenesis, tumorigenesis, and cell-cycle progression. The effect of MDM2 on these processes depends, in part, on its ability to inactivate p53 tumor suppressor gene.[16] The role of MDM2 in regulation of p53 stability highlights its primary role in regulation of normal growth and in the prevention of cellular transformation which results in abnormal growth and tumorigenesis when deregulated.[15]

MDM2 itself is the product of p53-inducible gene. Thus, the two molecules are linked to each other through autoregulatory negative feedback loop aimed at maintaining low cellular p53 levels in the absence of stress. Principally, MDM2 is an E3 ligase and promotes p53 degradation through an ubiquitin-dependent pathway on nuclear and cytoplasmic 26S proteasomes. Protein modification by ubiquitin conjugation is a general intracellular targeting mechanism and covalently attached polyubiquitin chains on lysine residues target proteins to proteasomes for degradation.[17]

The present study was undertaken in order to provide a scientific basis for differences noted in biologic behavior between variants of ameloblastoma and OKC based on their diverse proliferative potentialities.

The results obtained from the quantitative analysis of the labeling index of p53 in SMA, OKC, and UA [Table 1] indicate the proliferating activity of OKC which is minimum in UA. This observation is in accordance with the studies done by Piattelli et al.,[18] de Oliveira et al.,[19] and Sharifi-Sistani et al.[20] in 2011 where they studied the immunoexpression of p53 and MDM2 in ameloblastoma and OKC, and the increased expression of p53 was seen in OKC followed by SMA and UA. This could be attributed to the fact that the epithelial lining of OKC contains more cells in the cell cycle than in other types of odontogenic cysts. This concept was put forward by Browne[21] (1994). Contradictorily, Carvalhais et al.[8] have investigated p53 and MDM2 expression in KOT, ameloblastoma, radicular cyst, and adenomatoid OT and found no increased expression of p53 and its positive correlation with MDM2 [Table 2].

Moreover, p53 positivity was seen in 14 cases of SMA which is in agreement with the study done by el-Sissy and Al-Fayad where they stated that positive staining of p53 in SMA indicates disturbed growth regulation due to the existence of certain stimuli which triggered neoplastic transformation.[22],[23] It was further stated that detectable expression of p53 proteins may reflect stabilization of the protein via the interaction with other intercellular proteins. However, the expression of p53 in head-and-neck carcinomas stated that the level of p53 is often elevated in tumors derived from transformed cell lines and suggested that the inactivation of p53 gene confers the selective advantage for the development of the tumorigenic phenotype with its subsequent impact on changing cellular activity (Krishna et al. 2012) and Maass et al.[24],[25]

Immunopositivity of p53 in cases of UA indicates a probable reason of its neoplastic transformation which otherwise shows benign course and low recurrence rate. These results of our study are in accordance with the study done by el-Sissy (1999) and Al-Fayad indicating that p53 abnormalities play a crucial role in early neoplastic transformation during the development of UA.[22],[23]

Of 20 cases of each group, 30%, 3%, and 60%, of cases are of SMA, OKC, and UA, respectively, which are immunonegative for p53. The possible reasons for immunonegativity of p53 expression could be due to: first, lack of mutation – wild-type p53 protein has a short half-life and is undetectable; second, the mutation did not result in stabilization of protein; and third, p53 gene was detectable.[8] The p53 forms an autoregulatory feedback loop with MDM2, and MDM2, in turn, negatively regulates p53 and inhibits its function as transcription factor and causes degradation of intracelluar p53.[4]

The results of post hoc comparison of p53 immunoexpression in the three study groups indicating a benign course of UA with low-level expression in nontransformed cell and act as a negative regulator of cell division due to the fact that, the wild-type protein p53 does not normally accumulate to a detectable amount because of a short half-life (6–20 min).[23] The greater number of p53 positive cell in OKC and SMA indicates a similarity in their biological behavior and their activity in the same direction.[20]

Further, the immunoexpression of p53 in the basal and parabasal layers of all the three study groups was seen, and the results showed that the expression of p53 in the basal layer is maximum in SMA (18.15 ± 12.351), and in the suprabasal layer, p53 expression was maximum in OKC. The results were statistically significant (P< 0.00). The probable reason of these results in SMA demonstrated that basal cells have high proliferative activity and are responsible for neoplastic transformation which may be triggered by p53 overexpression possible as a defense mechanism to perform its tumor suppressor function (el-Sissy). Further, Browne (1994) stated that a unique proliferative and differentiation process occurs in the cystic lining of OKC. The presence of differentiated cells in the basal cell layer accounts for the fact that the major proliferative compartment is suprabasal.[21] The epithelial lining of OKC contains more cells in the cell cycle predominantly in suprabasal cells than that in other types of odontogenic cysts which reflected by the increased number of mitotic figures and higher mitotic index in the suprabasal cell layer[22],[23] [Table 2].

Statistically significant results of labeling index of MDM2 in SMA, OKC, and UA [Table 3] were seen which show that the expression of MDM2 is maximum in SMA (37.50 ± 20.37) followed by OKC (46.70 ± 16.91) and was minimum in UA (23.00 ± 19.43) which indicates the proliferative activity of different types of ameloblastoma and OKC. On contrary to our results, Sharifi-Sistani et al. also studied MDM2 expression in ameloblastoma and OKC, and their result shows no significant difference among variants of ameloblastoma and OKC.[21] However, Carvalhais et al. (1999) and Krishna et al. have investigated MDM2 expression in odontogenic cyst and tumor and their results show positive expression of MDM2 in OT and cyst. The reason for the positive expression of MDM2 is associated with cell-cycle progression and apoptosis suggesting that MDM2 plays a central role in cancer development and progression.[8],[24]{Table 3}

The p53-MDM2 paradigm represents the best-studied relationship between a tumor suppressor gene and an oncogene. These two genes form an autoregulatory feedback loop where MDM2 inhibiting p53 functions as transcription factor and also inducing intracellular p53 degradation. It has been demonstrated that single-nucleotide polymorphism 309, resulting in a T to G alteration within the first intron of the MDM2 gene promoter which can enhance MDM2 expression and therefore lead to a reduced tumor suppressor function of p53 and overproduction of MDM2 protein which gives rise to a greater risk in the development of malignancy Krishna et al.[24] According to Carvalhais, et al. the overexpressed MDM2 protein exerts its own p53-independent oncogenic activity and contributes to neoplastic development as MDM2 protein interacts with other cellular proteins involved in cycle regulation including pRB, E2F1/DP1, and p19 Alternate reading frame (ARF).[8]

Further, in our study, the post hoc comparison of MDM2 labeling index was done in the three study groups which shows statistically significant difference among the mean number of positive cells in SMA and UA and OKC and UA, whereas the comparison of mean number of positive cells of SMA with OKC was nonsignificant. These results are in accordance with the study of Krishna et al. 2012, where they have seen a higher percentage of positive cells in SMA.[24] These results are further elucidated by Kumamoto et al. where they described increased apoptosis associated with high reactivity for caspase 3 and FAS in UA, indicating UA as a low-grade tumor with low proliferative index. It also supports the contention of lower recurrence rate in simple UA as compared to SMA.[8]

On comparing the immunoexpression of MDM2 in the basal and parabasal layers of all the three study groups, expression of MDM2 in basal layer is maximum in SMA (30.20 ± 11.04) and in suprabasal layer MDM2 expression was maximum in OKC (26.45 ± 14.12). The results were statistically significant (P< 0.00) [Table 3]. The probable reason for the variation of these expressions in the basal and the parabasal layers may be because of the expression of MDM2 reactivity mainly in peripheral columnar or cuboidal cells and occasionally in central stellate reticulum-like cells, suggesting that the peripheral cells have an antiapoptotic and proliferative phenotype unlike the central cells.[24]

Qualitatively, higher staining intensity of p53 was seen in OKC and SMA; these results were supported by Slootweg where he postulated that it was the overexpression of p53 rather than increased number of p53 positive cells that were related to the proliferative capacity of OKC and SMA.[26] Immunohistochemical presence of p53 protein indicates a disturbance of growth regulation. Hence, altered p53 metabolism, whether due to mutation or due to changed turnover of the wild-type protein, is related to cell proliferation.

Due to the above-mentioned reason, the negative or mild staining intensity of p53 in UA can be put forth and supported by the study done by Kumamoto et al.[27] As in UA, there is an increased number of normally acting p53 positive cells which is of wild type and is not stable to be accumulated to a particular concentration to provide intense p53 staining, as they undergo destruction by p53-MDM2 pathway.

The qualitative analysis of MDM2 in the study group in OKC shows intermediate (40%) to high (30%) intensity; in SMA, half of the cases show high (50%) intensity; and in UA, it is intermediate (25%) to high (20%) intensity. This result is in contraindication with the result of Sharifi-Sistani et al. where they found moderate intensity in all the three groups, i.e., SMA, OKC, and UA, but in our study, SMA shows high intensity than OKC and UA.[20]

In our study, we observed that p53 and MDM2 are positively correlated. The increased expression of p53 and MDM2 protein plays a role in pathogenesis and tumorigenesis of SMA, OKC, and UA. This increased expression is probably an explanatory mechanism for the similarities in biological behavior of these odontogenic lesions and their activity in the same direction. As it is stated by Hao and Cho and Moll and Petrenko in normal unstressed cells, p53 is very unstable protein with a half-life ranging from 5 to 30 min, which is present at very low cellular levels owing to continuous degradation largely mediated by MDM2. Three possible mechanisms of p53 degradation by MDM2: first, abrogate activity of p53 by MDM2 binding to its N-terminal transactivation domain; second, it may destabilize the p53 by promoting its poly-ubiquitination and proteasomal degradation; and finally, it promotes nuclear export of p53 by inducing monoubiquitination. Conversely, a hallmark of many cellular stress pathways such as DNA damage, hypoxia, telomere shortening, and oncogene activation causes rapid stabilization of p53 by phosphorylation of p53 and MDM2, thus inhibiting the interaction of p53 with MDM2 as it becomes stable and further deficient in activating MDM2 resulting in the defective negative feedback loop, therefore, resulting in the increased expression of p53 and MDM2 in odontogenic lesions.[17],[28]


 > Conclusion Top


It is concluded that positive correlation of p53-MDM2 relationship is vital which helps in determining the pathogenetic model and tumorigenic nature of SMA, OKC, and UA which is probably an explanatory mechanism for the indistinguishability in biologic behavior of these lesions and their activity in the same direction. Thus, this two-way relationship between MDM2 and p53 forms an autoregulatory negative feedback and has a significant role in etiology and progression of neoplasms and therefore is an important topic for future research which should help in the development of new therapeutics against cancer.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

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