It is well established that oral carcinogenesis derives from progression of preinvasive neoplastic lesions that are characterized by grade specific morphological alterations . Viruses have been implicated in malignant neoplasia of squamous epithelia so it is conceivable that they might contribute aetiologically in some cases of oral carcinoma .
Although overexpression of EBV is considered to contribute to oral carcinogenesis, there is few literatures supporting this hypothesis [34, 35] and several reports described the up-regulation of EBV expression in dysplasia and carcinoma of the head and neck cancer [34, 36]. Therefore, the present study was designed to investigate the immunohistochemical expression of EBV in the 38 cases of oral epithelial dysplasia and oral squamous cell carcinoma and thereby to elucidate the involvement of EBV in oral carcinogenesis. In the present study, pattern of EBV immunostaining in the normal control sections revealed negative results. This coincides with the results of Talacko et al  and Shimakage et al  who found no detectable EBV DNA in oral mucosa of normal individuals using in situ hybridization technique. They suggested that EBV replicates upon entry into the oral terminally differentiated keratinocytes rather than remaining latent.
Our study showed that 10 out 16 cases (62.5%) of epithelial dysplasia were positive for anti-EBV antibody. This is in agreement with Cruz and co-workers  who reported the presence of EBV in 77.8% of premalignant lesions. In contrast, Naher et al  and Horiuchi et al  found that EBV genome was detected in 8.6% and 5.3% of their studied oral leukoplakia cases respectively.
An interesting finding was noted in the present study. The EBV genome was detected in the middle and upper layers of squamous epithelium more than in the basal cell layer. This result agrees with Greenspan  and Sandvei  who demonstrated the presence of EBV receptors in the prickle layer of normal oral epithelium and oral hairy leukoplakia.
In the current study, 18 out of the 22 (81.8%) examined squamous cell carcinomas were positive for anti-EBV antibody. This is in agreement with Mao and Smith  and Horiuchi et al  who showed that 35% and 33.3% of their examined squamous cell carcinoma cases respectively were infected with EBV. They stated that the positively rate was higher in malignant lesions than in benign ones and agreed that EBV had a role in carcinogenesis; however, it may exist in cancer cells as a passenger. A higher percentage was noticed by Van Rensburg , Cruz and Co-workers . They observed EBV in 100% of the oral squamous cell carcinomas, reflecting difference in analytical methods as they used PCR which is a highly sensitive technique.
Contradictory to our results, Talacko et al  using insitu-hybridization demonstrated that EBV DNA was not present in the neoplastic cells of oral squamous cell carcinoma. They concluded that there was no evidence for a role of EBV in the process of malignant transformation of oral mucosa in immunocompetent individuals.
In the present work, immunoreactive EBV was detected within the keratin pearls and in the central malignant cells of squamous cell carcinoma. The pattern of staining was nuclear, perinuclear, cytoplasmic as well as total cell reactivity. This is in agreement with the findings of Greenspan , Horiuchi et al , and Sandvej  who confirmed the presence of EBV in a similar pattern of reaction.
Thus in this study, the presence of EBV proteins was demonstrated in (73.6%) of all our studied cases while negative results were observed in the control specimens. This result implies a possible direct relation between EBV infection and epithelial dysplasia as well as squamous cell carcinoma. Accordingly, our study appears to support the view of others concerning the role of EBV in carcinogenesis [34, 35].
In the present study, cancerous lesions of the oral mucosa expressed EBV more prominent than did oral epithelial dysplasia lesions. EBV expression was increased as tumor differentiation was decreased.
In our study, we found that increased age did not enhance EBV prevalence. Thus, the age does not seem to be a risk factor for EBV infection. This result was in accordance with other reports .
The relationship of proliferation markers with the grading of dysplasia is uncertain, and the present investigation is an attempt to remedy this. Recent immunohistochemical studies have shown Topo II α to be a reliable indicator of cell proliferation in tumors, such as breast, ovarian, and bladder carcinomas, vulvar squamous lesions and oral lichen planus [42–46]. Here, Topo II α was immunohistochemically assessed in oral epithelial dysplasias, and carcinomas. Topo II α is an essential cellular enzyme that functions in the segregation of newly replicated chromosome pairs, in chromosome condensation, and in altering DNA superhelicity . Because the expression of Topo II α isoform increases during the late S phase, decreases at the end of the M phase, and is dramatically reduced in the G1/G0 phase of the cell cycle,  an anti-Topo II α antibody labels cells in the S, G2, and M phases of the cell cycle . The immunohistochemical method for the in situ determination of Topo II α has been validated extensively and shown to reflect closely the exact enzyme activity in formalin fixed paraffin wax embedded human tissues . On the contrary, techniques such as western or northern blotting are averaging techniques and do not assess the amount of Topo II α in one particular neoplastic cell.
In general, the biological behavior of human malignancies is influenced by two major biological features of cancer cells: abnormal proliferation and the potential to invade and metastasise, which occasionally correlate with each other .
In the present study, the intense positive nuclear immunostaining expression of Topo II α was observed in the basal and parabasal layers (stratum germnativum layer) of nearly all normal oral epithelium regions while the granular and corneum cell layer showed less and variable degree of positive nuclear immunostaining. This is in agreement with the results of Liu et al.,  who studied the proliferative rate in intra oral sites and found that basal cell layer and parabasal layer (stratum germnativum cell layer) show high proliferative activity and no proliferative activity was seen above the parabasal layer (superficial layer). In this field Holden et al , Hellemans et al  and Sandri et al  explained that the distribution of Topo II α in normal human tissue was restricted to sites known to harbor actively dividing cells. Also Truly et al  found that Topo II α is detected in proliferating compartment of all normal tissues, as would be expected with a cell division-specific role, as mitotic chromosomes segregation and/or condensation.
Hotchin, Gandarillas and Watt  found that the proliferation of keratinocyte is primarily restricted to the basal layer. When keratinocytes divide, down-regulate cell surface integrins to lose adhesiveness, leave the basal layer, exit the cell cycle and undergo a program of terminal maturation as they move through the suprabasal layers to the tissue surface. During this journey, the keratinocytes undergo a series of physiological and morphological changes that terminate by production of dead, flattened enucleated squames that are shed and replenished by differentiating keratinocytes.
The intense nuclear and cytoplasmic immunoexpression of Topo II α is observed in basal and parabasal layer in studied epithelial tissues, although Earnshaw et al  revealed that Topo II α has been shown to be a component of two highly insoluble protein fractions from chromosomes and nuclei, so these observation suggested that Topo II α might be an integral structure of the nucleus.
The Topo II α in the studied tissues gives variable reaction among different human oral tissues. The values of Topo II α between studied cases cleared that it can be ordered the oral tissues from highest to lowest immunoexpression as follow lip, gingival inferior surface of tongue and hard palate. The difference in immunoexpression of Topo II α among different normal oral human tissues may be explained by different opinion of many authors, Kellett, Hume and Potten, , who studied the proliferation rates and DNA synthesis in a continuous strip of epithelium from the gingival sulcus to the ventral surface of the tongue of mice and they found that the peak of proliferation and DNA synthesis in floor of mouth (lining mucosa) is higher followed by attached gingiva then free gingival epithelium. They concluded that there are various aspects of the distribution of DNA synthesis and proliferation in relation to type and site of the studied cells.
The epithelial covering of the labial mucous membrane in the present study showed highest nuclear immunoexpression of Topo II α among different other oral tissue. This is coincided with Thomson et al  who studied the difference in mitotic index between human oral mucosa tissues and found that there was a significant difference between anatomical sites, with higher mitotic count observed in buccal mucosa than mandibular gingiva. Meanwhile Nagase et al  stated that the turnover time is the estimated time needed to replace all cells in the epithelium and is derived from knowledge of time taken for a certain cell to divided and pass through the entire epithelium. They added that the nonkeratinized epithelium turnover faster than keratinized gingival epithelium and in general the turnover time for skin needs 75 days, for cheek 25 days and for gingiva 41–57 days. They concluded that there are regional difference in a pattern of epithelial proliferation and maturation associated with different turnover rates.
In the present study the keratinized epithelium of the gingival tissues showed higher expression to Topo II α than keratinized epithelium of hard palate in spite of that both tissues are masticatory mucosa, this may be due to the difference in type of keratinization as Eckert, Crish and Robinson  stated that in the oral cavity, Orthokeratinized epithelium similar to that in skin is seen in the hard palate, whereas other regions are either parakeratinized (gingiva) and parakeratinized epithelium divide faster than Orthokeratinized epithelium.
Also in the present study the epithelium of the lining mucosa of the lip showed higher immunostaining than that found in inferior surface of tongue this may be explained by Burns et al  explained that there are several possible explanations the most obvious explanation relates to the possibility that gene expression may be regulated differently. And the regional differences in gene expression could have many possible consequences. For example, differential responsiveness to hormones and growth factors and in their receptor, as there are differences in alpha adrenergic receptor number, might in turn influence the time of appearance and activity of enzymes and regulators involved in replication and differentiation.
In more details Philippe et al  suggested that the differences in behavior of epithelial tissue from various regions may reflect on site intrinsic characteristics to cells, or cell clones rather than to external factor. They explained that the disparate properties intrinsic to cells might due to regionally different modes of cellular communication including direct transmission of chemical messengers between contiguous cells either by paracrine and autocrine mechanisms. They added that regional differences in adipose tissue growth and distribution reflect intrinsic cellular properties, rather than locational disparities or external influences as blood or nerve supply, or ambient temperature.
In the current work the epithelial lining of oral mucous membrane of young group reveal intense immunoexpression to Topo II α than old group of different human oral tissues population, they are highly significant P < 0.01. This is in agreement with Stewart et al , who demonstrated that the cells associated with aging, is decreased metabolic efficiency (reduced growth rate), reduced off spring production, and with increased chance of death. Cardelli et al.  found that a progressive decrease of proliferation rate was found during both physiologic aging in vivo and induced aging in vitro.
In the other hand, Celenligil-Nazliel et al  found that there is no significant difference was observed between different age group with respect to proliferative activity in healthy gingiva. Age-related changes in proliferative activity in human gingival epithelium are uncertain. All the tissue sections contained positive staining cells for PCNA in the gingival epithelium. Although PCNA expression was observed both in the basal and suprabasal layers, it was more prominent in the suprabasal layers. But in inflamed gingiva was significantly higher in the older group. The proliferative activity was found to be increased with aging.
As well as Evans, Galasko and Ward,  studied the effect of age on growth of bone and found that the ability of individual cells to divide and to perform specific synthetic activities namely, total protein, osteocalcin, and alkaline phosphatase synthesis, did not show any change with increasing donor age. These results suggest that while the ability of individual cells to divide and to perform is unimpaired with increasing age, other subtler changes may occur, leading to a decrease in the bone's osteogenic capacity.
In our study, we also found that the Topo II-α index increased with progression from OED to OSCC, presumably reflecting the increase in the number of cycling tumor cells in invasive carcinomas.
Topo II-α was related to the clinicopathological parameters and no significant correlation was found between Topo II-α expression and patient's sex and tumor site. This finding was in agreement with previous reports on oral squamous cell carcinoma .
With regard to the age of OSCCs. High Topo II-α expression was more frequently detected among young age group of OSCCs and the correlation of high Topo II-α expression and age was statistically significant. In contrast, Stathopoulos et al  found that there was no statistically significant correlation between them.
With regard to the degree of tumour differentiation, the incidence of high Topo II-α immunopositivity was significantly greater in poorly differentiated SCCs. This strong correlation is in agreement with one previous study in head and neck squamous cell carcinomas  as well as in other malignancies [49, 52]. In malignant cells, overexpression of the Topo II-α protein might reflect not only the proliferative advantage of these cells, but also qualitative alterations caused by malignant transformation and dedifferentiation.  In a recent study, treatment of the hepatoma cell line Hep3B with retinoids(which can induce cell differentiation) appeared to have a direct effect on the Topo II-α gene promoter because it greatly reduced both the steady state amount of mRNA and the transcription rate of the Topo II-α gene. This Topo II-α activity increased as tumor differentiation was decreased and primary tumor size (T), regional lymph node metastasis (N), and disease stage advanced, resulting in poor prognosis. This finding was in agreement with Segawa et al  who carried out a study on oral squamous cell carcinomas. On the other hand, Stathopoulos et al  reported differently and stated that Topo II-α index of head and neck squamous cell carcinoma did not correlate with clinicopathological parameters such as histologic type, lymph node metastsasis, and tumor site. In addition, we found that there was significant relationship between Topo II-α and EBV expression in primary lesions was higher in cases with lymph node metastasis than in those without lymph node metastasis. As EBV expression increased, the Topo II-α LI significantly increased. These findings coincided with those of the present study. Therefore, these two enzyme activities may be valuable diagnostic and prognostic indices in oral carcinoma.
As regards the two evaluated markers (Topo II-α and EBV), they were not significantly related to each other in OSCCs. However, increased expression of EBV correlated significantly with elevated Topo II-α in OEDs. In addition to, the expression of EBV was increased in the progression from of OEDs to OSCCs, with elevation of Topo II-α LI. These findings strongly indicate that EBV may contribute to malignant transformation and tumor growth. This result was in agreement with Shimakage .