Title: Analysis of Bcl-2 and p53 protein expression in non-Hodgkin's lymphoma
Abstract: The growth of the lymphoid cells is regulated by a delicate balance between molecules controlling cell survival and cell death. The Bcl-2 gene product is an anti-apoptotic molecule that modulates the mitochondrial release of cytochrome c, and the interaction of Apoptosis activating factors with caspase 9 and Bax (Bcl-2 associated × protein). p53 is a tumor suppressor gene that maintains genomic stability either by inducing cell cycle arrest or apoptosis. Although some studies examined p53 and bcl-2 protein expression in non-Hodgkin's lymphoma (NHL), side-by-side analysis of these proteins in the different grades of NHL is still lacking [1.Du M. Singh N. Husseuin A. Isaacson P.G. et al.Positive correlation between apoptotic and proliferative indices in gastrointestinal lymphomas of mucosa-associated lymphoid tissue (MALT).J Pathol. 1996; 178: 379-384Crossref PubMed Scopus (56) Google Scholar, 2.Gisbertz I.A. Schouten H.C. Bot F.J. et al.Proliferation and apoptosis in primary gastric B-cell non-Hodgkin's lymphoma.Histopathology. 1997; 30: 152-159Crossref PubMed Scopus (20) Google Scholar, 3.Charalambous G.K. Gomatos I.P. Konstadoulakis M.M. et al.Protein expression of bax, bcl-2, and p53 in patients with non-Hodgkin's gastric lymphoma: prognostic significance.World J Surg. 2000; 24: 608-614Crossref PubMed Scopus (16) Google Scholar]. To address this issue, formalin-fixed, paraffin-embedded tissue specimens representing 47 cases of NHL were obtained from the Departments of Pathology, Assuit University Hospitals. Immunohistochemical evaluation was carried out using immunoperoxidase staining methods and mouse monoclonal antibodies (clones 124, IgG1, kappa, and DO1 for Bcl-2 and p53, respectively; DAKO Corporation, Denmark). Additional sections, running in parallel but with omission of the primary antibodies, served as the negative controls. The positive controls consisted of lymph nodes with reactive hyperplasia (for bcl-2) and squamous cell carcinoma (for p53). The percentages of positive cells (PP%) of p53 (nuclear staining) and bcl-2 (cytoplasmic staining) protein expression, apoptotic (AI) and mitotic (MI) indices were evaluated [4.Chan W.Y. Cheung K.K. Schorge J.O. et al.Bcl-2 and p53 protein expression, apoptosis, and p53 mutation in human epithelial ovarian cancers.Am J Pathol. 2000; 156: 409-417Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar]. Our results revealed gradual down-regulation of bcl-2 staining values with the transition from low to intermediate- to high-grade NHL (87.7 ± 4.9 > 72.60 ± 3.4 > 66.1 ± 3.3, P=0.041). This down-regulation may be due to (i) up-regulation of bcl-2 antagonists, i.e. p53 protein, (ii) another prosurvival molecule rather than Bcl-2 protein takes over its role and (iii) epigenetic mechanisms such as Bcl-2 promoter hypermethylation [5.Xu W. Sheng R. Sheng Z. Xu T. et al.The expression and clinical significance of proliferative antigen Ki-67 and apoptosis-antagonizing antigen Bcl-2 in non-Hodgkin's lymphoma.Zhonghua Nei Ke Za Zhi. 2001; 40: 452-455PubMed Google Scholar]. Alternatively, the p53 staining values showed gradual up-regulation with the transition from low to intermediate to-high-grade NHL (6.50 ± 3.1 < 12.8 ± 4.8 < 18.5 ± 5.1, P=0.023). This up-regulation may be due to the presence of p53 gene mutations that stabilize the mutant p53 protein, and increase its half-life leading to its accumulation in the cells [3.Charalambous G.K. Gomatos I.P. Konstadoulakis M.M. et al.Protein expression of bax, bcl-2, and p53 in patients with non-Hodgkin's gastric lymphoma: prognostic significance.World J Surg. 2000; 24: 608-614Crossref PubMed Scopus (16) Google Scholar]. The negative correlation between bcl-2 and p53 protein expression in NHL (r=−0.221, P=0.165) suggests that the former is negatively regulated by the latter (Figure 1). The increase in the AI and MI with the transition from low (2.60 ± 0.4 and 2.0 ± 0.3) → intermediate (5.2 ± 0.5 and 5.1 ± 0.5) → high (7.1 ± 0.6 and 6.9 ± 0.4) grade NHL is in agreement with previous studies [1.Du M. Singh N. Husseuin A. Isaacson P.G. et al.Positive correlation between apoptotic and proliferative indices in gastrointestinal lymphomas of mucosa-associated lymphoid tissue (MALT).J Pathol. 1996; 178: 379-384Crossref PubMed Scopus (56) Google Scholar, 2.Gisbertz I.A. Schouten H.C. Bot F.J. et al.Proliferation and apoptosis in primary gastric B-cell non-Hodgkin's lymphoma.Histopathology. 1997; 30: 152-159Crossref PubMed Scopus (20) Google Scholar, 3.Charalambous G.K. Gomatos I.P. Konstadoulakis M.M. et al.Protein expression of bax, bcl-2, and p53 in patients with non-Hodgkin's gastric lymphoma: prognostic significance.World J Surg. 2000; 24: 608-614Crossref PubMed Scopus (16) Google Scholar] (Figure 1). The increased MI may be due to inactivation of p53 gene → loss of its inhibitory effects on its downstream effector genes → promotion of cell cycle progression and cellular proliferation. Alternatively, the increased AI may be due to (i) down-regulation of bcl-2 and (ii) induction of p53 independent apoptosis due to activation of another oncogene such as c-Myc that takes over the role of inactivated p53 in apoptosis [1.Du M. Singh N. Husseuin A. Isaacson P.G. et al.Positive correlation between apoptotic and proliferative indices in gastrointestinal lymphomas of mucosa-associated lymphoid tissue (MALT).J Pathol. 1996; 178: 379-384Crossref PubMed Scopus (56) Google Scholar]. In summary, our study proposes that altered bcl-2 and p53 protein expression is involved in lymphomagenesis.