INTRODUCTION
Cyclin-dependent kinases (CDKs) regulate the progression of the cell cycle1). The CDKs phophorylate the retinoblastoma susceptibility gene protein which then allows the progression of the cell cycle from G1 into the S-phase1–3). The CDKs are activated by phosphorylation by activating CDK-activating kinases. The CDKs inhibitors block this activation of CDKs by CDK-activating kinases. The inhibition of CDK activation results in the inhibition of retinoblastoma susceptibility gene phosphorylation and, consequently, in cell cycle arrest in the G1 phase1–3). Therefore, the CDK inhibitors have been regarded as putative tumor suppressors. The CDK inhibitors can be considered as two distinct groups of enzymes. Group 1 is Cip/Kip family, including p21, p27 and p574–8). Group 2 is INK family including p15, p16, p18 and p199–12). Among the many reported CDK inhibitors, decreased expression of p21 and p27 has been described in neoplastic cells and has also been associated with tumor progression and poor outcome in various human cancers, including gastric cancer13–20).
The p53 tumor suppressor gene is believed to play a pivotal role in preventing the uncontrolled cellular growth characteristic of cancer. p53 is mutated or deleted in about 50% of spontaneously arising tumors and this alteration of p53 is strongly associated with tumor progression and metastasis21–24). Recently, it has been reported that the expression of p21 is induced by the tumor suppressor gene p534, 7, 25, 26). Thus, the function of p21 as a tumor suppressor may be not retained after p53 alteration in human cancers.
The purpose of our study was to evaluate the prognostic significance of these tumor biomarkers as tumor suppressors relative to the information derived from established clinicopathological variables in gastric cancers.
MATERIALS AND METHODS
Patients and tumor specimens
One hundred and two patients who underwent surgery for gastric cancer from July 1992 to June 1993 at Chonnam National University Hospital, Gwangju, Korea were selected retrospectively for this study. The patients’ ages ranged between 28 and 79 years (mean, 58.4). 65 were male and 37 were female. The size of tumors ranged between 0.5 and 15.0 cm (mean, 5.1). No patient had received chemotherapy or radiation therapy before surgery. The tumors were staged at the time of surgery by the standard criteria for TNM staging used by the American Joint Committee on Cancer27). Patient characteristics including sex, age, histologic grade, stage and survival data, were obtained from medical records and pathologist and physician contact when necessary. Survival was measured until follow-up at June 2002. The clinicopathological characteristics of the study populations are summarized in Table 1.
Imunohistochemistry
Immunohistochemical staining was performed by the Micro-Probe staining system (Fisher Scientific, Pittsburgh, PA) based on capillary action28). Paraffin sections, of 4 μm in thickness with mounted probe on slides, were immunostained with anti-mouse monoclonal antibodies by the avidin-biotin peroxidase complex method28). Sections were deparaffinized and heated in a microwave oven for 7 minutes to retrieve the antigens. They were immersed in 0.6% hydrogen peroxide for 5 minutes to block the endogenous peroxidase activity. The primary antibodies used were a mouse monoclonal antibody against human p21 protein (Santa Cruz Biotechnology, Santa Cruz, Calf. USA) in a 1:200 dilution and human p53 protein (Dakopatts, Glostrup, Denmark) in a 1:100. The primary antibodies, in the aforementioned concentrations were diluted in phosphate-buffered saline supplemented with 5% normal horse serum and 1% bovine serum albumin and then incubated with tissues for 120 minutes at room temperature. Anti-mouse immunoglobulin G (Sigma, St. Louis, MO) labeled with biotin was added as a secondary antibody for the detection of primary antibodies and the samples were incubated for 7 minutes at 45°C. After multiple rinses with universal buffer, streptavidin-alkaline phosphatase detection system (Biomeda, Foster, CA) was applied for 7 minutes. As the final step, the slides were developed for 20 minutes with the enzyme substrate 3 amino-9-ethyl carbazole (AEC, Sigma, St. Louis, MO). The slides were counterstained with hematoxylin solution for 1 minute (Research Genetics, Huntsville, AL). After dehydration, the tissue was sealed with a universal mount (Research Genetics, Huntsville, AL). Normal immunoglobulin G was substituted for each primary antibody as negative control.
Scoring of p21 and p53 expression
According to a previous report, p21 and p53 immunoreactivity was assessed as being positive only when tumors exhibited intense nuclear staining, and reactivity was categorized into 2 groups: negative expression (less than 10% positive tumor cells) and positive expression (at least 10% positive tumor cells)29, 30). Assessment of immunoreactivity was evaluated by two independent observers without knowledge of the corresponding clinicopathological data.
Statistical analysis
The χ2-test and Fisher’s exact test, where appropriate, were used to compare the expression of p21 and p53 with various clinicopathological variables. Survival curves were calculated using the Kaplan-Meier method and analyzed by the log-rank test. The statistical software program used was Statistical Package for the Social Sciences (SPSS/PC+ 10.0, Chicago, IL). Findings of p<0.05 were taken to indicate statistical significance.
DISCUSSION
Tumor formation and growth are characterized by uncontrolled cellular proliferation. This is usually the result of multiple genetic and epigenetic insults to the cell, particularly involving proto-oncogenes and tumor suppressor genes31).
The dysfunction of p53, a tumor suppressor gene, has been regarded as the most significant event in the pathogenesis of cancer. Mutation of p53 lost its suppressor function and also gained new function which altered the phenotypic features of tumor cells. Mutation of p53 has been investigated in a wide variety of human cancers with a view to correlating possible loss of its suppressor function with tumor development, progression and prognosis21–24).
In our study, positive expression of p53 correlated with the depth of tumor invasion. However, there was no apparent association between survival and expression of p53. It is interesting to note that conflicting data exist regarding the predictive power of p53 status with regard to survival. These contradictory findings might be due to the methods used to establish p53 status, patient population size and the heterogeneity in the surgical treatment provided to these patients. Also, previous reports have shown discordance rates when compared with techniques that determine gene status32). Thus, the expression of p53 as detected by immunohistochemistry is not an accurate measurement of p53 function.
In recent years, alterations of the genes encoding such cell cycle regulators, as well as oncogenes and tumor suppressor genes, have been reported to contribute to carcinogenesis33). Deregulation of cyclin, CDKs and their inhibitors could have an important role in many types of human cancers1–3). Several CDK inhibitors have been identified and potentially act as tumor suppressors. Among the many reported CDK inhibitors, it has been reported that the expression of CDK inhibitor p21 is associated with tumor suppression13, 14, 19). Our study showed that negative expression of p21 correlated with the advanced stage, lymph node metastasis and poor survival. These results suggest that p21 expression may be a useful marker of prognosis. However, other studies reported that there is no significant correlation between p21 expression and the various clinicopathological parameters, including survival34, 35).
p53-induced cell growth arrest is due to the potential of p53 to regulate one or more cell cycle check point-related genes, which include MDM2, Gadd45 and p214, 7, 25, 26). Of these targets of p53, the p21 gene is the primary mediator of p53-induced cell cycle arrest. However, in our study, there was no correlation between p53 and p21 expression. Gomyo et al. and Noda et al. reported that no significant correlation is noted between p21 and p53 expression13, 29). Also, Michieli et al. reported that serum or individual growth factors, such as platelet-derived growth factor, fibroblast growth factor and epidermal growth factor are able to induce p21 in quiescent p53-deficient cells, as well as in normal cells36). Therefore, our results and other reports suggest that induction of p21 expression can occur via p53 independent pathways in a considerable proportion of carcinomas. Further studies are warranted to clarify the impact of p53 on the function of p21 as a tumor suppressor.
Previous reports showed that the combined analysis of p21 and p53 expression yielded more accurate prognostic information30, 37). In our study, combined analysis of p21 and p53 status also showed that p21 negative and p53 positive tumors had poorer survival than other group tumors. Our result is similar with previous reports. These results suggest that tumor progression and prognosis are not dependent on single tumor suppressor gene alteration and are regulated by multiple genetic and epigenetic insults to the cell, particularly involving tumor suppressor genes.
In conclusion, the status of p21 and p53 expression may help in predicting the aggressive behavior of gastric cancer. However, further studies are warranted to clarify the impact of p53 on the function of p21 as a tumor suppressor.