Esophageal carcinoma is an age-related neoplasm with a 5-year overall survival rate of less than 35% (1,2). Males more than 40 years of age are at the highest risk of esophageal carcinoma (3). Results of studies have revealed that age-related changes affect the molecular crosstalk between the stromal and epithelial cells, generating a more permissive environment for tumor growth and metastasis (4). Senescence may be one of the mechanism which is responsible for this age-related change in the microenvironment. The definition of senescence is a process that keeps the stable form of cell cycle arrest at the G₁ phase (5), as first reported by Hayflick (6). At the earlier stage of the process, senescence acts as an antiproliferative factor, which prevents tumorigenesis and leads to cell cycle arrest. However, if oncogenic mutations are present, these senescent cells may become immortal and initiate the development of additional genetic ‘hits’ in tumorigenesis (7–9). Cell cycle regulators within the retinoblastoma (Rb) and p53 pathways, including the cyclin-dependent kinase (CDK) and CDK inhibitor (CDKI) proteins, are the gatekeepers which maintain the senescence program. p14^ARF, p15^INK4b and p16^INK4a have been identified as CDKIs and act as senescence maintenance factors.

The INK4a/ARF locus (9p21) is crucial in the pathogenesis of several types of malignant disorders and encodes two unrelated cell cycle regulators, p14^ARF and p16^INK4a, which have been regarded as significant senescence markers in clinical diagnosis (10). The p16^INK4a gene encodes a p16 protein that binds competitively to CDK4 and, during G₁ phase, inhibits the interaction of CDK4 and cyclin D₁ to stimulate passage through the cell cycle (11–13). The p16 protein is often highly expressed in senescent cells in culture and is inactivated in a variety of human cancers. p15^INK4b is located centromeric to the p16/p14 gene locus p14^ARF, which is a major tumor suppressor and causes cell cycle arrest through transforming growth factor β (14).

Previous data revealed, through staining, that p14^ARF, p15^INK4b and p16^INK4a are abundantly expressed in premalignant lung cancers and have essentially no expression in malignant lung cancers, which indicates that senescence is inactivated in the proliferated lung epithelial cells of lung cancers (15). The same results have been observed in pancreatic, hepatic and breast cancers. Tumorigenesis is defined as an outcome of the accumulation of abnormal stroma facilitated by peripheral senescent fibroblasts and the inactivation of the senescence of proliferating epithelial cells. However, more recent evidence has revealed the high expression of the senescence markers p14^ARF, p15^INK4b, p16^INK4a and DCR2 in prostate cancer epithelial cells, indicating that senescence continued to be activated in these proliferating epithelial cells.

A balance between proliferation and senescence, instead of the inactivity of senescence only, appears to decide the fate of epithelial cells, converging on the probability of epithelial tumorigenesis.

Since the true mechanism involved in the process from senescence to tumor formation in epithelial cells remains elusive (16–19), in the present study we compared the expression of the senescence markers p14^ARF, p15^INK4b and p16^INK4a and the proliferation markers bcl-2, ki-67 and skp2 in tissue blocks from normal esophageal epithelium, esophageal intraepithelial dysplasia (EID) and esophageal squamous cell carcinoma (ESCC). The purpose of the present study was to describe the predictors of biological activity in esophageal carcinoma and to provide evidence that may assist in clinical diagnosis.

As shown in Fig. 1, p14^ARF, p15^INK4b, p16^INK4a and bcl-2 expression was observed in the cytoplasm of esophageal epithelial cells with no evidence of nuclear staining, while skp2 and ki-67 showed predominant nuclear staining at different portion of epithelial.

As shown in Table II, overall, p14^ARF, p15^INK4b and p16^INK4a showed almost complete negative expression in the normal esophageal epithelium, while marked positive expression was observed in the EID tissues and different pathological lesions of the ESCC tissues. More specifically, the p14^ARF expression was negative in most of the normal esophageal tissues (85%), but diffuse positive staining was observed in the EID (82%) and ESCC (100%) tissues, including 9 cases of EID and 60 cases of ESCC. The p16^INK4a staining was similar to that of p15^INK4b, with only 2 cases of normal esophageal tissue having weakly positive and most of the EID and ESCC tissues showing marked staining (73 and 88%, respectively). p15^INK4b showed positive staining in 73% of the EID and 92% of the ESCC specimens, while no positive staining was observed in the normal esophageal tissue specimens.

A total of 46 male and 14 female ESCC patients participated in this study, 53% of whom had lymph node metastasis. The TNM stages of these patients are listed in Table I. As shown in Table III, a statistically significant correlation was observed between p16^INK4a expression and the degree of differentiation. The poorly differentiated ESCC showed stronger p16^INK4a staining compared with the well-differentiated ESCC (P<0.05). p16^INK4a expression was observed in 95% of the poorly differentiated ESCC and only 71% of the well-differentiated ESCC tissues. The positive expression of p15^INK4b was observed in 95% cases of poorly differentiated ESCC and 82% cases of well-differentiated ESCC. The positive staining of p14^ARF was found in all the ESCC cases. Although there is no statistical evidence to verify that cases with lymph mode metastasis had a more marked immunostaining of p14^ARF, p15^INK4b and p16^INK4a, the positive staining ratios are high in these specimens. There was no statistical correlation between the expression of p14^ARF, p15^INK4b and p16^INK4a between the genders and among the TNM stages.

As shown in Table IV, there was positive staining for the proliferation marker skp2 in 64% of the EID and 72% of the ESCC cases but only 10% of the normal esophageal tissues. The analysis of ki-67 revealed a markedly high level of expression in the EID (91%) and ESCC (100%) cases, while there was a low incidence of positive staining in the basal epithelial cells of the normal tissue (20%). The bcl-2 immunostaining revealed that 36 ESCC cases (60%) and only 2 (10%) of the normal esophageal tissues had positive bcl-2 expression. As shown in Table V, cells that showed a positive expression of bcl-2 were found in 15 (88%) of the well-differentiated and 21 (49%) of the poorly differentiated ESCC cases. The variant expression of bcl-2 may be due to the heterogeneity of the cells.

The incidence of esophageal carcinoma rises exponentially with age, beginning from the fourth decade of life and peaking at the age of 75 years. Evidence suggests that cellular senescence is involved in causing organismal aging. Senescence, the process by which cells permanently withdraw from the cell cycle in response to diverse stress, may also contribute to this age-related disease (23). The senescence marker p16 was found to be upregulated with age in the progenitor cells of the mouse brain, bone marrow and pancreas. However, cellular senescence has two roles in cells. On one hand, senescence is associated with aging, which significantly increases the transformation of epithelial tumors. On the other hand, it is acknowledged that senescence is crucial in the prevention of cancer, which is achieved by inducing reversible proliferative arrest mediated by ARF/p53 and irreversible proliferative arrest mediated by the concomitant actions of INK4a/Rb and the p53 pathway (4,24). In this process, the main barrier to the overgrowth of cancer is the derepression of the INK4a/ARF locus (25,26). Overall, cellular senescence has two roles: cancer protection in the young and age promotion in the old. To the best of our knowledge, epithelial cells are the origin of most carcinomas, resulting from either the mutation of an oncogene in an epithelial cell or, in a paracrine manner, by an adjacent senescent fibroblast cell, converging on the promotion of epithelial tumorigenesis. However, whether senescence occurs in epithelial cells during transformation is unknown.

To date, evidence suggests that p14^ARF, p15^INK4b and p16^INK4a are widely downregulated in several solid tumors (27), including hepatic (28), breast, urinary bladder, pancreatic and esophageal carcinomas and gliomas (29). The INK4a/ARF locus encodes two significant tumor suppressors, p16^INK4 and ARF, which share the same exons but encode different reading frames. p16^INK4a is an inhibitor of CDK4 and CDK6 and acts by imposing a G₁ cell cycle arrest. The INK4a/ARF locus has been regarded as a controller of cancer evolution which is expressed at low levels in most tissues in young organisms but becomes derepressed with age. The inactivation of senescence markers, due to homozygous deletion or hypermethylation of the genes which encode them, may be a significant mechanism in the dysfunction of the Rb and p53 growth regulation pathways during ESCC development (30). However, it has been reported that the senescence pathway remains intact in a large number of prostate cancer and cervical squamous carcinoma cases (31) even if the ki-67 index indicates increased proliferation in these cancers. Zhang et al found that the senescence markers p14^ARF, p15^INK4b, p16^INK4a and DCR2 were expressed more frequently in prostate carcinomas than in benign tissues (32) and Meng et al identified activated senescence markers in colon cancer cells (33). Moreover, Schwarze identified an alteration in the p16/pRb pathway in the majority of primary prostate cancers in vitro (34). In our ESCC samples, the expression of p14^ARF, p15^INK4b and p16^INK4a increased during ESCC progression and was also associated with greater age, poor differentiation and, to some degree, a high tumor stage. That is, although cancer cells generally lose their ability to undergo senescence and apoptosis, certain tumor cells trigger senescence in response to severe DNA damage or other stimuli (16,35). Therefore, senescence may play a role in cancer development.

skp2 is a member of the Skp1-Cullin-F-box protein (SCF) complex and considered to be a proto-oncogene, as its overexpression causes increased proliferation and metastasis, at least in part through increased p27 proteolysis (24). Wang et al found that phosphorylation at Ser72 is crucial for the ability of the skp2 protein to promote cell proliferation and tumorigenesis, through several complementary mechanisms (36). skp2 also induces cells to undergo a mitotic division cycle by degrading p27 in early G₁ (37). The results of our immunostaining assays revealed that skp2 is activated at the dysplasia stage of esophageal tumorigenesis and maintains a high level of expression in most ESCCs (38). The uninhibited proliferation eventually leads to the development of ESCC.

The nuclear antigen ki-67, which is markedly expressed in the S and M phases of the cell cycle (39–41), was used to estimating cell growth. bcl-2 is a proto-oncogene which has been identified as a biologically significant inhibitor of apoptosis, whose overexpression leads to cell proliferation (42–44). In our data, a high expression of ki-67 was associated with the high expression of skp2 and the low expression of bcl-2, which acts as a marker of proliferation (27,45). We detected the expression of bcl-2 in EID and ESCC tissues and half of the ESCC cases showed a paradoxical loss of bcl-2, which indicated a poor prognosis. The high expression of bcl-2 in EID showed its anti-apoptotic function by blocking p53-mediated G₁ arrest (46). The paradoxical loss of bcl-2 in ESCC may not be explained as a result of chemo-radiotherapy, since none of the cases in our database were treated prior to surgery. However, the same results were obtained in cervical and endothelial carcinoma, indicating that other mechanisms may be involved.

In general, the role of senescence in tumorigenesis attracts a great deal of attention, particularly concerning the functions of p14^ARF, p15^INK4b and p16^INK4a. According to previous studies, tumorigenesis in old age not only reflects the accumulation of oncogenic mutations but also stromal alteration (47). Campisi (48) addresses these issues as good citizen and bad neighbors. The epithelial cells in squamous carcinomas always show senescence and apoptosis. Senescence serves as a powerful barrier for tumorigenesis in epithelial cells. However, in ESCC, the high expression of p14^ARF, p15^INK4b and p16^INK4a provides evidence for the activation of senescence in the epithelial rather than stromal cells. Further studies are required to explain this phenomenon. However, the expression of markers of senescence and proliferation, including p14^ARF, p15^INK4b, p16^INK4a, bcl-2, skp2 and ki-67 in ESCC and promalignancies and a loss of their expression in normal tissue and neoplasm may augment routine histological diagnostic methods for difficult cases.