Immunohistochemical Expression of p53, Bcl-2, Bax, and Fas Proteins in Squamous Cell Carcinomas From Immunosuppressed Renal Transplant Recipients And Immunocompetent Individuals D. Sec¸kin, B. Demirhan, H. Karakayalı, S. Akgu¨n, R. Erdal, and M. Turan
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ENAL transplant recipients (RTRs) are highly susceptible to malignant cutaneous tumors, particularly squamous cell carcinoma (SCC).1–3 Although many etiological factors (ultraviolet radiation, decreased cell-mediated immunity, chemical carcinogens, and human papillomavirus infections) have been suggested to contribute to the development of these lesions, the associated genetic abnormalities are not fully understood. Cutaneous SCCs in RTRs tend to show more aggressive biological behavior,4 metastasizing more frequently than those that arise in solardamaged skin of immunocompetent individuals. Current evidence suggests that a failure of the apoptotic response is critical to the development of skin cancers in the general population.5 It is possible that the molecular steps of this response may differ in transplant-associated and nontransplant-associated cutaneous carcinogenesis. We examined the immunohistochemical expression of some apoptosis regulators, namely, the p53, Bcl-2, Bax, and Fas proteins, in cutaneous SCCs and adjacent skin in immunosuppressed RTRs and immunocompetent individuals. The aim was to explain the different biological behavior of SCCs observed in immunosuppressed RTRs. PATIENTS AND METHODS Formalin-fixed paraffin-embedded sections of 10 cutaneous SCCs from sun-exposed sites in RTRs and 14 cutaneous SCCs from sun-exposed sites in immunocompetent patients were examined. Standard avidin-biotin-peroxidase immunostaining techniques were used. The primary antibodies were anti-p53 monoclonal antibody, anti-Bcl-2 monoclonal antibody, anti-Bax polyclonal antibody, and anti-APO-1/Fas monoclonal antibody (DAKO, Calif, USA). For negative controls, we omitted the primary antibody. Immunoreactivity was characterized using a semiquantitative scoring system that has been described previously.6 Statistical analysis was performed using Fisher’s exact chi-square test.
RESULTS
Expression of p53, Bcl-2, Bax, and Fas was observed in 8 (80%), 1 (10%), 5 (50%), and 3 (30%) of the tumors from the RTRs, respectively. The same proteins were present in 10 (71.4%), 2 (14.3%), 4 (28.6%), and 3 (21.4%) of the SCCs from the immunocompetent patients, respectively. © 2002 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 34, 2139 –2140 (2002)
The differences between the groups were not statistically significant (P ⬎ .05). We found that p53 immunoreactivity was mostly confined to the tumor margins, with gradual loss of staining in the more differentiated areas. We were able to examine normal skin adjacent to the tumoral tissue in 7 of the 10 RTRs and in all the control patients. In the RTR group, we noted expression of p53, Bcl-2, Bax, and Fas in 2 (28.6%), 1 (14.3%), 0 (0%), and 4 (57.1%) patients, respectively. The same proteins were detected in 9 (64.3%), 3 (21.4%), 2 (14.3%), and 7 (50%) of the immunocompetent patients, respectively. Expression of the proteins in the adjacent skin was similar in both groups (P ⬎ .05). DISCUSSION
Apoptosis is a programmed or physiological form of cell death that was first described by Kerr et al7 in 1972. An active and genetically controlled process, apoptosis is considered to play a critical role in the proliferation of various neoplasms because the rate of tumor growth depends on the balance between tumor cell proliferation and tumor cell death. Disruption of the apoptotic response has also been implicated in various skin cancers.5 The p53, Bcl-2, Bax, and Fas proteins are pivotal participants in the regulatory mechanisms of apoptosis. p53 has been called the guardian of the genome and causes cellcycle arrest at the G1 phase until DNA damage has been repaired.8 This function is lost in approximately 50% of human tumors, where p53 is inactivated by a mutation in the gene that encodes it or by the binding proteins encoded by viral or cellular oncogenes.9 Mutated p53 accumulates in the nuclei of damaged cells but fails to mediate tumor suppression.8 Bcl-2 is a protein of the inner mitochondrial membrane that has been shown to suppress apoptosis by binding Bax.5,10 Overexpression of Bcl-2 has been reported in several malignant tumors. When the Bax protein itself is overexpressed, it allows apoptosis to proceed. Bcl-2 and Bax From Bas¸kent University Faculty of Medicine, Ankara, Turkey. Address reprint requests to D. Sec¸kin, Bas¸kent University Faculty of Medicine, 1. Cadde No:77 Bahcelievler, Ankara 06490, Turkey. E-mail:
[email protected] 0041-1345/02/$–see front matter PII S0041-1345(02)02882-8 2139
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coordinate to control apoptosis.5,10 Fas (Apo-1/CD95), a cell surface receptor and a member of the tumor necrosis factor superfamily, induces apoptosis after oligomerization by its ligand (FasL). Recent studies have suggested that defects within the Fas receptor pathway play an important role in the development and progression of human tumors.5 Several studies have investigated the role of p53 gene mutation or p53 protein inactivation in cutaneous SCCs in RTRs.11–17 To our knowledge, however, expression of the Bcl-2, Bax, and Fas proteins has not been studied previously. The results of p53 studies vary, probably due to the tumor samples chosen and the techniques and antibodies used. Khorshid et al11 found less p53 immunoreactivity in SCCs from transplant recipients than in nontransplant SCCs. They suggested that mechanisms other than p53 gene mutation might be important in the pathogenesis of posttransplantation skin cancers. Results of other studies have indicated no difference in p53 immunoreactivity between SCCs from RTRs and those from nonimmunosuppressed individuals.12–14 A different study has also shown that the frequency and type of p53 mutations in transplant SCCs are similar to the findings in sporadic SCCs.15 However, the study by Ferra´ndiz et al16 noted higher prevalence and more extensive p53 immunoreactivity in dysplastic epidermal keratoses of less than 3 years’ duration in RTRs compared with similar lesions in the general population. In light of this, these authors proposed that p53 alterations in RTRs are associated with tumor initiation or promotion. The high prevalence of p53 immunoreactivity that we observed in SCCs from immunosuppressed and nonimmunosuppressed patients provides more evidence that p53 gene mutation or p53 protein inactivation plays an essential role in the pathogenesis of these tumors. However, consistent with the results of some previous studies,12–14 we found no difference in the overall prevalence of p53 immunostain-
SEC¸ KIN, DEMIRHAN, KARAKAYALI ET AL
ing in transplant- and nontransplant associated SCCs and adjacent skin. Moreover, we also found no differences in the levels of expression of other important apoptosis regulators (Bcl-2, Bax, and Fas proteins) in the 2 groups. Although this is a preliminary study and the number of cases is limited, it appears likely that factors other than alteration of the apoptotic process underlie the high prevalence and more aggressive behavior of SCCs in RTRs. REFERENCES 1. Lugo-Janer G, Sanchez JL, Santiago-Delpin E: J Am Acad Dermatol 24:410, 1991 2. Sec¸kin D, Oguz Gu ¨lec¸ T, Demirag˘ A, et al: Transplant Proc 30:802, 1998 3. Ferra´ndiz C, Fuente MJ, Ribera M, et al: J Am Acad Dermatol 33:590, 1995 4. Barba A, Tessari G, Boschiero L, et al: Nephron 73:131, 1996 5. Teraki Y, Shiohara T: Eur J Dermatol 9:413, 1999 6. Oram Y, Orengo I, Baer SC, et al: J Am Acad Dermatol 31:417, 1994 7. Kerr JF, Wyllie AH, Currie AR: Br J Cancer 26:239, 1972 8. Harris CC: Science 262:1980, 1993 9. Greenblatt MS, Bennett WP, Hollstein M, et al: Cancer Res 54:4855, 1994 10. Korsmeyer SJ, Shuttler JR, Veis DJ, et al: Semin Cancer Biol 4:327, 1993 11. Khorshid SM, Glover MT, Churchill L, et al: J Cutan Pathol 23:229, 1996 12. McGregor JM, Farthing A, Crook T, et al: J Am Acad Dermatol 30:701, 1994 13. Gibson GE, O’Grady A, Kay EW, et al: J Am Acad Dermatol 36:924, 1997 14. Stark LA, Arends MJ, McLaren KM, et al: Br J Cancer 70:662, 1994 15. McGregor JM, Berkhout RJM, Rozycka M, et al: Oncogene 15:1737, 1997 16. Ferra´ndiz C, Fuente MJ, Ferna´ndez-Figueras MT, et al: Dermatol Surg 25:97, 1999 17. Bennett MA, O’Grady A, Kay EW, et al: Biochem Soc Trans 25:342, 1997
