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p73 Protein Expression Correlates With Radiation-Induced Apoptosis in the Lack of p53 Response to Radiation Therapy for Cervical Cancer
Int. J. Radiation Oncology Biol. Phys., Vol. 70, No. 4, pp. 1189–1194, 2008 Copyright Ó 2008 Elsevier Inc. Printed in the USA. All rights reserved 0360-3016/08/$–see front matter
doi:10.1016/j.ijrobp.2007.08.033
CLINICAL INVESTIGATION
Cervix
P73 PROTEIN EXPRESSION CORRELATES WITH RADIATION-INDUCED APOPTOSIS IN THE LACK OF P53 RESPONSE TO RADIATION THERAPY FOR CERVICAL CANCER MASARU WAKATSUKI, M.D.,*y TATSUYA OHNO, M.D., PH.D.,y MAYUMI IWAKAWA, M.D., PH.D.,y HITOSHI ISHIKAWA, M.D., PH.D.,* SHUHEI NODA, M.D., PH.D.,y TOSHIE OHTA, M.S.,y SHINGO KATO, M.D., PH.D.,y HIROHIKO TSUJII, M.D., PH.D.,y TAKASHI IMAI, M.D., PH.D.,y AND TAKASHI NAKANO, M.D., PH.D.* * Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan; and y Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, Chiba, Japan Purpose: p73 belongs to the p53 tumor suppressor family of genes and can inhibit cell growth in a p53-like manner by inducing apoptosis or cell cycle arrest. Here, we investigated whether p73 could compensate for impaired p53 function in apoptosis induced by radiation therapy (RT) for cervical cancer. Methods and Materials: Sixty-eight patients with squamous cell carcinoma of the cervix who received definitive RT combined with (n = 37) or without (n = 31) cisplatin were investigated. Biopsy specimens were excised from the cervical tumor before RT and after 9 Gy. Results: Mean apoptosis index (AI) was 0.93% before RT and 1.97% after 9 Gy with a significant increase (p < 0.001). For all patients, there was a significant correlation between p73 expression positivity after 9 Gy and AI ratio (AI after 9 Gy/AI before RT) (p = 0.021). Forty-one patients were regarded as the p53-responding group according to the expression of p53 after 9 Gy, whereas the remaining 27 patients were regarded as the p53–nonresponding group. A significant correlation between p73 expression after 9 Gy and AI ratio was observed in the p53-non-responding group (p < 0.001) but not in the p53-responding group (p = 0.940). Conclusion: Our results suggest that p73 plays an important role in compensating for the lack of p53 function in radiation-induced apoptosis of cervical cancer. Ó 2008 Elsevier Inc. Cervical cancer, Radiation therapy, Apoptosis, p73, p53.
The efficacy of radiation therapy (RT) in cervical cancer is well-established. Recently, however, cisplatin-based chemoradiation therapy (CRT) for locally advanced cervical cancer has shown a 30–50% survival benefit over RT alone (1). Despite this therapeutic innovation, pelvic recurrence was observed in 18–27% of patients even after CRT, and improvement of pelvic control is still needed (2, 3). Apoptosis, which means programmed cell death, is an active model of cell death that occurs in response to DNA damage by ionizing radiation, ultraviolet irradiation, and certain chemotherapeutic agents (4, 5). A large number of experimental studies have revealed that apoptosis induced by irradiation is a determining factor of radiosensitivity (6–9). Further, in RT for cervical cancer, a high occurrence of apoptotic cells after a total dose of 9 Gy was significantly associated with better pelvic control (10).
p73 belongs to the p53 tumor suppressor family of genes and shares significant homology with the p53 structural organization (11). Overexpression of p73 induced by irradiation activates the transcription of p53-responsive genes such as p21, Bax, Mdm2, and GADD45, and inhibits cell growth in a p53-like manner via the induction of apoptosis or cell-cycle arrest (12–15). In addition, p73 protein appears to be able to induce apoptosis independently of p53 in vitro (16, 17). However, it is unclear whether these mechanisms of apoptosis underlying p73 are also involved in the clinical effect of RT in cervical cancer (18). Many investigators have shown the impact of p53 status on treatment outcomes in patients with cervical cancer (6–8). Our previous study demonstrated that p53 mutation significantly worsened the local control of cervical cancer treated with RT (9). It is well known that E6 protein produced by human papillomavirus infection, which is associated with the development of cervical cancer, inactivates the normal
Reprint requests to: Tatsuya Ohno, MD, PhD, Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan. Tel: (+81) 43-206-3360; Fax: (+81) 43-256-6506; E-mail: [email protected] Supported by the National Institute of Radiological Sciences.
Conflict of interest: none. Acknowledgment—We thank M. Sakai for assisting with the TUNEL assay and immunohistochemistry. Received Nov 14, 2006, and in revised form May 30, 2007. Accepted for publication Aug 5, 2007.
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function of p53 (19). However, because the E6 protein does not inactivate p73 function, it would be interesting to know whether p73 can compensate for the impaired p53 function to trigger the apoptosis of cervical cancer cells in response to radiation (20). Here, we investigated the relation between p73 expression and radiation-induced apoptosis, with particular emphasis on whether p73 can compensate for impaired p53 function in the apoptosis induced by RT for cervical cancer. MATERIALS AND METHODS Patients Tumor samples obtained from 68 patients with cervical cancer who received RT alone or CRT at the National Institute of Radiological Sciences between 2002 and 2005 were investigated. All tumors were confirmed histologically as squamous cell carcinoma. Clinical stage was determined according to the classification of the International Federation of Gynecology and Obstetrics (21). The number of patients with Stage I, II, III, and IV disease was 5, 11, 37, and 15, respectively. Patient age ranged from 37 to 83 years, with a mean of 58 years. The study was approved by the Institutional Review Board of the National Institute of Radiological Sciences, and written informed consent was obtained from each patient before entry.
Treatment For RT, all patients were treated with a combination of external beam and high-dose-rate intracavitary irradiation. The standard treatment method for cervical cancer in our institute has been described previously (22). In brief, external irradiation to the whole pelvis was performed with anteroposterior and posteroanterior parallel opposed ports with a total dose of 30.6 Gy at 1.8 Gy per fraction, five times per week. This was followed by a central shielding pelvis field up to a total pelvis irradiation dose of 50.6 Gy at 2 Gy per fraction, five times per week. Along with the central shielding irradiation, intracavitary irradiation by a remote afterloading system using an iridium-192 source was performed. Four fractions of intracavitary irradiation were administered once per week at a fraction dose of 5–7 Gy at Point A, with the total dose ranging from 23 to 28 Gy. Among the 68 patients, 37 received CRT, which consisted of cisplatin at 40 mg/m2 weekly starting from week 1 for five consecutive weeks during the course of external beam irradiation. Eligibility criteria for CRT were as follows: Stage IIb >4 cm in diameter or Stage IIIb or IV disease; age between 20 and 70 years; performance status of 0–2; and adequate baseline bone marrow, hepatic, and renal function (white blood cell $3,000/mm3, hemoglobin $10 g/dL, platelet $100,000/mm3, total bilirubin #1.5 mg/dL, L-aspartate aminotransferase (AST)/L-alanine aminotransferase (ALT) #2 times the upper normal limit, and serum creatinine #1.5 mg/dL). Patients were excluded from CRT if they met any of the following criteria: prior chemotherapy or RT for the pelvis, severe pelvic infection, or severe psychological illness. All punch biopsy specimens were excised from the nonnecrotic cervical tumors before RT and after a total dose of 9 Gy (five fractions was given) for RT alone or 9 Gy plus one course of cisplatin for CRT. The second biopsy was taken from the same tumor area. All tissues were first fixed in 10% formalin for 1 day, embedded in paraffin, and sectioned into 3-mm serial sections. One section was stained with hematoxylin and eosin and the rest were subjected to the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method (TUNEL) assay for apoptotic cell defec-
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tion and immunohistochemical staining for p53 and p73 proteins. Tumor cell content was independently reviewed by a pathologist.
TUNEL assay Apoptotic cells were detected using the ApopTag Peroxidase in situ Apoptosis Detection kit (Chemicon International, Temecula, CA). The automated function of the Ventana Discovery System (Ventana Medical Systems, Tucson, AZ) was used from deparaffinization to counterstaining without titration of terminal deoxynucleotidyl transferase enzyme linkage of dUTP-digoxigenin to 30 -OH DNA ends. Briefly, formalin-fixed, paraffin-embedded sections (3 mm thick) from biopsy specimens were deparaffinized for 8 min by EZ Prep (Ventana Medical Systems). Protease 1 (Ventana Medical Systems) was treated for 30 min at 37 C. A mixture of ApopTag terminal deoxynucleotidyl transferase enzyme and ApopTag reaction buffer was applied for 40 min at 37 C, and then the reaction was stopped with ApopTag stop/wash buffer applied for 30 min at 37 C. Secondary antibody (monoclonal anti-DIGOXIN biotin conjugate; Sigma-Aldrich, St. Louis, MO) at a 1:500 dilution was applied for 52 min at 37 C. Finally, 3,3-diaminobenzidine (DAB) was used to detect the bound antibody complex. Slides were counterstained with hematoxylin. Apoptotic cells were identified on the basis of both TUNEL assay and characteristic morphological changes identified by hematoxylin and eosin staining, such as chromatin condensation and blebbing of the nucleus (Fig. 1). Artifact areas of nuclear staining associated with massive cellular necrosis were excluded from the analysis. The apoptotic cell index (AI) was calculated by dividing positive cells by the number of more than 2000 tumor cells in randomly selected fields in each case under a light microscope. In addition, AI ratio (AI after 9 Gy divided by AI before RT) was calculated to detect treatment-related apoptosis, and used to separate patients into high (AI ratio >2) and low AI ratio groups (AI ratio #2).
Immunohistochemistry Immunohistochemical studies were performed to detect the expressions of p53 and p73 proteins using a standard DAB-based immunostaining procedure employing a DAB Map Kit (Ventana Medical Systems). Sections were deparaffinized and cell
Fig. 1. Apoptotic cells stained by dUTP-biotin nick end labeling. Tumor cells with chromatin condensation and blebbing of the nucleus are positively stained, showing apoptotic cells (original magnification 400).
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Fig. 2. Immunohistochemical staining for p53 (A) and p73 (B) proteins. Immunostaining showed p53 (A) and p73 (B) positivity in the nucleus of tumor cells (original magnification 400). conditioning was performed (60 mins, 37 C) using the same automated Ventana system as above. Antibodies for p53 DO7 (Cell Marque, Hot Spring, AR) and p73 (Neomarkers, Fremont, CA) were diluted 100:1 and 50:1 in antibody dilution buffer (Ventana Medical Systems), respectively. Slides were incubated in the solution for 40 min. They were then incubated for 30 min with a universal secondary antibody (Ventana Medical Systems), counterstained with hematoxylin and postcounterstained with bluing reagent. Slides with high levels of p53 and p73 immunoreactivity were used as positive controls. Negative controls, without the primary antibody, were incubated in each step. Random sampling of more than 2,000 cells per tumor evaluated in three or four microscopic fields was then done. Positive staining for p53 or p73 protein was defined when more than 10% of tumor cells were stained with these proteins in each section (23, 24). In addition, in accordance with the staining response of p53 after 9 Gy of irradiation, samples were divided into a p53responding (p53 after 9 Gy/p53 before RT >1, and expression of p53 after 9 Gy >10%) and a p53–nonresponding group (p53 after 9 Gy/p53 before RT #1, or expression of p53 after 9 Gy #10%).
Statistical analysis Differences between AI before RT and AI after a total dose of 9 Gy were determined by means of the paired Student’s t-test. The unpaired Student’s t-test was used for the differences among AI, p53, and p73 positivity according to age group (>60 years vs. #60 years), stage (I and II vs. III and IV), and treatment (RT vs. CRT). The chisquare test was used for statistical analysis of the correlation between p53 expression or p73 expression and AI ratio. Fisher’s exact test was used for statistical analysis of the correlation between p73 expression after 9 Gy and AI ratio in the p53–nonresponding group. A p value of less than 0.05 was considered significant.
RESULTS Apoptotic index The mean AI was 0.93% (range, 0.02–3.06%) before irradiation and 1.97% (range, 0.26–4.83%) after 9 Gy, indicating a significant increase in AI in response to RT (p < 0.001). The high AI ratio group accounted for 40 (59%) of 68 patients and the low AI ratio group the other 28 (41%). Age (>60 years vs. #60 years), stage (I and II vs. III and IV), and combination of
chemotherapy were not associated with AI before RT or after 9 Gy. p53 and p73 expression p53 staining was seen in the nucleus of tumor cells (Fig. 2A). Thirty-four (50%) of 68 patients had positive p53 expression before RT and 44 (65%) were positive after 9 Gy, which was not a significant increase (p = 0.083). Based on our criteria, 41 (60%) patients were classified into the p53responding group and the remaining 27 (40%) into the p53– nonresponding group. Twenty-one (51%) of 41 patients received CRT in the p53-responding group, and 16 (59%) of 27 patients received CRT in the p53–nonresponding group. p73 staining was seen in the nucleus of tumor cells (Fig. 2B). Positive p73 expression was observed in 48 (71%) patients before RT and in 38 (56%) after 9 Gy. The difference in p73-positive rates before and after RT was not significant (p = 0.075), and no significant correlation between p53 and p73 expression before RT was seen (Table 1, p = 0.110). p53 expression positivity before RT was 19.5% for early stage (Stage I and II) and 10.2% for advanced stage (Stage III and IV) disease, with this difference being significant (p = 0.04). p73 expression positivity after 9 Gy was 9.4% for early stage (Stage I and II) and 16.9% for advanced stage (Stage III and IV) cancer, which was again significant (p = 0.03). Age (>60 years vs. #60 years) and combination
Table 1. Correlation between p53 and p73 expression before RT p53 before RT
p73 before RT
+ –
+
–
27 7
21 13
Abbreviation: RT = radiation therapy.
p = 0.110
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Table 2. Correlation between p53 or p73 expression before RT and AI ratio for all patients p53 before RT
AI ratio*
High(>2) Low(#2)
p73 before RT
+
–
20 14
20 14
p > 0.999
+
–
32 16
8 12
p = 0.042
Abbreviations: RT = radiation therapy; AI = apoptotic index. * Apoptotic index after 9 Gy/apoptotic index before RT.
of chemotherapy were not associated with p53 expression before RT and after 9 Gy, nor with p73 expression before RT and after 9 Gy. Correlation between apoptotic index and p53 or p73 expression As shown in Table 2, there was no significant correlation between p53 expression positivity before RT and AI ratio (p > 0.999). In contrast, a significant correlation between p73 expression positivity and AI ratio was observed before RT for all 68 patients (p = 0.042). As shown in Table 3, significant correlations were seen between p53 (p = 0.038) or p73 expression positivity (p = 0.021) after 9 Gy and AI ratio for all patients. In the p53-responding group, no significant correlation was seen between p73 expression after 9 Gy and AI ratio (p = 0.940, Table 4). In contrast, a significant positive correlation was seen between p73 expression after 9 Gy and AI ratio in the p53–nonresponding group (p < 0.001, Table 4). DISCUSSION In the present study, p73 expression positivity was 71% before RT. Singh et al. reported a positivity ratio before treatment of cervical cancer of 42% (19). Given that p73 positivity significantly increased with advancing disease stage in ovarian cancer, one reason for our higher positivity than that of Singh et al. may be differences in the proportion of patients with advanced disease (19, 25), with 76% of our patients having Stage III-IV disease vs. only 28% in their study. The current study showed no significant increase in p73 expression positivity after RT at 9 Gy, but did show a significant increase in that for p53. Several studies have reported irradiation-induced p73 expression in various tumor cells in vitro. Among them, Pucci et al. showed that p73 expression was induced by irradiation in glioblastoma cells lacking
the p53 gene (26). Lin et al. also showed that p73 levels were increased by irradiation in both p53 null mouse fibroblasts and p53 null human osteosarcoma cells (27). These studies suggested that p73 might be induced in cancer cells in which p53 function was absent. Further, several studies revealed that p73 was downregulated by p53 activation in breast cancer and lung cancer cells (28, 29). We therefore speculate that one reason that p73 expression did not significantly increase after 9 Gy was derived from a p53-induced negative feedback mechanism. The correlation of p73 expression with apoptosis in cervical cancer treated with RT remains unclear. Although p73 induces apoptosis by a different mechanism to p53 (30), it shares target genes with p53 (14, 31, 32). In the present study, no significant correlation was seen between p53 expression before RT and AI ratio, whereas p73 expression before RT correlated with AI ratio (p = 0.042). Further, a positive correlation was seen between p73 expression after 9 Gy and AI ratio, particularly in the p53–nonresponding group (p < 0.001). Irwin et al. reported that the induction of apoptosis was blocked by the inhibition of p73-dependent transcriptional activation in osteosarcoma cells, with a loss of p53 function (18). Schmid et al. also reported that doxorubicin increased p73 expression and induced the apoptosis of embryo fibroblast cells in a p53-lacking mouse (33). These experimental studies support our hypothesis that p73 may compensate for p53 function in the induction of apoptosis after irradiation for cervical cancer, when p53 function is inactivated by human papillomavirus infection. Prognosis has been shown to be poor in p73-positive patients undergoing surgery for colorectal adenocarcinoma, ovarian cancer and hepatocellular carcinoma (32, 34, 35). In contrast, p73 overexpression before RT was significantly associated with better survival in patients with cervical cancer (36). Given that it is possibly influenced by different treatment modalities and types of diseases, the prognostic
Table 3. Correlation between p53 or p73 expression after 9 Gy and AI ratio for all patients p53 after 9 Gy
AI ratio*
High(>2) Low(#2)
p73 after 9 Gy
+
–
31 15
9 13
Abbreviations as in Table 2. * Apoptotic index after 9 Gy/apoptotic index before RT.
p = 0.038
+
–
27 11
13 17
p = 0.021
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Table 4. Correlation between p73 expression after 9 Gy and AI ratio in the p53-responding group or the p53–nonresponding group p53-responding group p73 after 9 Gy
AI ratio*
High(>2) Low(#2)
+
–
16 8
12 5
p53–nonresponding group p73 after 9 Gy
p = 0.940
+
–
11 3
1 12
p < 0.001
Abbreviations as in Table 2. * Apoptotic index after 9 Gy/apoptotic index before RT.
value of p73 expression remains controversial. The prognostic significance of p73 expression in the present study could not be analyzed owing to the short follow-up period. To summarize, we confirmed the occurrence of radiationinduced apoptosis in patients with cervical cancer treated with RT or CRT by TUNEL assay and morphologic findings,
and of p53 and p73 expression by immunohistochemical methods. A significant positive correlation between p73 expression after 9 Gy and AI ratio was observed in the p53–nonresponding group. These results suggest that p73 may play a key role in the induction of apoptosis in cervical cancer patients with impaired p53 function.
REFERENCES 1. Rose PG, Bundy BN. Chemoradiation for locally advanced cervical cancer: does it help? J Clin Oncol 2002;20:891–893. 2. Eifel PJ, Winter K, Morris M, et al. Pelvic irradiation with concurrent chemotherapy versus pelvic and para-aortic irradiation for high-risk cervical cancer: an update of radiation therapy oncology group trial (RTOG) 90-01. J Clin Oncol 2004;22: 872–880. 3. Pearcey R, Brundage M, Drouin P, et al. Phase III trial comparing radical radiotherapy with and without cisplatin chemotherapy in patients with advanced squamous cell cancer of the cervix. J Clin Oncol 2002;20:966–972. 4. Kerr JFR, Wylie AH, Currie AR. Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972;26:239–257. 5. Kerr JFR, Winterford CM, Harmon BV. Apoptosis: Its significance in cancer and cancer therapy. Cancer 1994;73: 2013–2016. 6. Oka K, Nakano T, Arai T. p53 CM1 expression is not associated with prognosis in uterine cervical carcinoma. Cancer 1993;72: 160–164. 7. Gitsch G, Kainz C, Joura E, et al. Mutant p53 product in patients with Stage III cervical cancer. Anticancer Res 1992;12: 2241–2242. 8. Crook T, Wrede D, Tidy J, et al. Clonal p53 mutation in primary cervical cancer: Association with human papilloma virus-negative tumors. Lancet 1992;339:1070–1073. 9. Ishikawa H, Mitsuhashi N, Sakurai H, et al. The effects of p53 status and human papillomavirus infection on the clinical outcome of patients with stage IIIB cervical carcinoma treated with radiation therapy alone. Cancer 2001;91:80–89. 10. Yuki H, Fujimura M, Yamakawa Y, et al. Detection of apoptosis and expression of apoptosis-associated proteins as early predictors of prognosis after irradiation therapy in stage IIIb uterine cervical cancer. Jpn J Cancer Res 2000;91:127–134. 11. Kaghad M, Bonnet H, Yang A, et al. Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers. Cell 1997;90:809–819. 12. Kaelin WG Jr. The p53 gene family. Oncogene 1999;18: 7701–7705. 13. Zhu J, Jiang J, Zhou W, et al. The potential tumor suppressor p73 differentially regulates cellular p53 target genes. Cancer Res 1998;58:5061–5065. 14. Cuddihy AR, Bristow RG. The p53 protein family and radiation sensitivity: Yes or no? Cancer Metastasis Rev 2004;23: 237–257.
15. Benard J, Douc-Rasy S, Ahomadegbe JC. TP53 family members and human cancers. Hum Mutat 2003;21:182–191. 16. Yang A, Kaghad M, Caput D, et al. On the shoulders of giants: p63, p73 and the rise of p53. Trends Genet 2002;18:90–95. 17. Jost CA, Marin MC, Kaelin WG Jr. p73 is a simian [correction of human] p53-related protein that can induce apoptosis. Nature 1997;389:191–194. 18. Irwin M, Marin MC, Phillips AC, et al. Role for the p53 homologue p73 in E2F-1-induced apoptosis. Nature 2000;407: 645–648. 19. Singh A, Sharma H, Salhan S, et al. Evaluation of expression of apoptosis-related proteins and their correlation with HPV, telomerase activity, and apoptotic index in cervical cancer. Pathobiology 2004;71:314–322. 20. Das S, El-Deiry WS, Somasundaram K. Efficient growth inhibition of HPV 16 E6-expressing cells by an adenovirus-expressing p53 homologue p73beta. Oncogene 2003;22:8394–8402. 21. Shepherd JH. Cervical and vulva cancer: Changes in FIGO definitions of staging. Br J Obstet Gynaecol 1996;103:405–406. 22. Nakano T, Kato S, Ohno T, et al. Long-term results of highdose rate intracavitary brachytherapy for squamous cell carcinoma of the uterine cervix. Cancer 2005;103:92–101. 23. Ishikawa H, Sakurai H, Hasegawa M, et al. Expression of hypoxic-inducible factor 1alpha predicts metastasis-free survival after radiation therapy alone in stage IIIB cervical squamous cell carcinoma. Int J Radiat Oncol Biol Phys 2004;60:513–521. 24. Chen YK, Hsue SS, Lin LM. p73 expression for human buccal epithelial dysplasia and squamous cell carcinoma: does it correlate with nodal status of carcinoma and is there a relationship with malignant change of epithelial dysplasia? Head Neck 2004;26:945–952. 25. Niyazi M, Ghazizadeh M, Konishi H, et al. Expression of p73 and c-Abl proteins in human ovarian carcinomas. J Nipp Med Sch 2003;70:234–242. 26. Pucci B, Claudio PP, Masciullo V, et al. pRb2/p130 promotes radiation-induced cell death in the glioblastoma cell line HJC12 by p73 upregulation and Bcl-2 downregulation. Oncogene 2002;21:5897–5905. 27. Lin KW, Nam SY, Toh WH, et al. Multiple stress signals induce p73beta accumulation. Neoplasia 2004;6:546–557. 28. Ohtsuka T, Ryu H, Minamishima YA, et al. Modulation of p53 and p73 levels by cyclin G: Implication of a negative feedback regulation. Oncogene 2003;22:1678–1687.
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29. Stiewe T, Theseling CC, Putzer BM. Transactivation-deficient DTA-p73 inhibits p53 by direct competition for DNA binding. J Biol Chem 2002;277:14177–14185. 30. Ramadan S, Terrinoni A, Catani MV, et al. p73 induces apoptosis by different mechanisms. Biochem Biophys Res Commun 2005;331:713–717. 31. Hamer G, Gademan IS, Kal HB, et al. Role for c-Abl and p73 in the radiation response of male germ cells. Oncogene 2001;20: 4298–4304. 32. Agami R, Blandino G, Oren M, et al. Interaction of c-Abl and p73alpha and their collaboration to induce apoptosis. Nature 1999;399:809–813. 33. Schmid G, Wang ZQ, Wesierska-Gadek J. Compensatory expression of p73 in PARP-deficient mouse fibroblasts as
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response to a reduced level of regularly spliced wild-type p53 protein. Biochem Biophys Res Commun 1999;255:399–405. 34. Tannapfel A, Engeland K, Weinans L, et al. Expression of p73, a novel protein related to the p53 tumour suppressor p53, and apoptosis in cholangiocellular carcinoma of the liver. Br J Cancer 1999;80:1069–1074. 35. Herath NI, Kew MC, Whitehall VL, et al. p73 is up-regulated in a subset of hepatocellular carcinoma. Hepatology 2000;31: 601–605. 36. Liu SS, Leung RC, Chan KY, et al. Related Articles, Links: p73 expression is associated with the cellular radiosensitivity in cervical cancer after radiotherapy. Clin Cancer Res 2004;10: 3309–3316.
doi:10.1016/j.ijrobp.2007.08.033
CLINICAL INVESTIGATION
Cervix
P73 PROTEIN EXPRESSION CORRELATES WITH RADIATION-INDUCED APOPTOSIS IN THE LACK OF P53 RESPONSE TO RADIATION THERAPY FOR CERVICAL CANCER MASARU WAKATSUKI, M.D.,*y TATSUYA OHNO, M.D., PH.D.,y MAYUMI IWAKAWA, M.D., PH.D.,y HITOSHI ISHIKAWA, M.D., PH.D.,* SHUHEI NODA, M.D., PH.D.,y TOSHIE OHTA, M.S.,y SHINGO KATO, M.D., PH.D.,y HIROHIKO TSUJII, M.D., PH.D.,y TAKASHI IMAI, M.D., PH.D.,y AND TAKASHI NAKANO, M.D., PH.D.* * Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan; and y Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, Chiba, Japan Purpose: p73 belongs to the p53 tumor suppressor family of genes and can inhibit cell growth in a p53-like manner by inducing apoptosis or cell cycle arrest. Here, we investigated whether p73 could compensate for impaired p53 function in apoptosis induced by radiation therapy (RT) for cervical cancer. Methods and Materials: Sixty-eight patients with squamous cell carcinoma of the cervix who received definitive RT combined with (n = 37) or without (n = 31) cisplatin were investigated. Biopsy specimens were excised from the cervical tumor before RT and after 9 Gy. Results: Mean apoptosis index (AI) was 0.93% before RT and 1.97% after 9 Gy with a significant increase (p < 0.001). For all patients, there was a significant correlation between p73 expression positivity after 9 Gy and AI ratio (AI after 9 Gy/AI before RT) (p = 0.021). Forty-one patients were regarded as the p53-responding group according to the expression of p53 after 9 Gy, whereas the remaining 27 patients were regarded as the p53–nonresponding group. A significant correlation between p73 expression after 9 Gy and AI ratio was observed in the p53-non-responding group (p < 0.001) but not in the p53-responding group (p = 0.940). Conclusion: Our results suggest that p73 plays an important role in compensating for the lack of p53 function in radiation-induced apoptosis of cervical cancer. Ó 2008 Elsevier Inc. Cervical cancer, Radiation therapy, Apoptosis, p73, p53.
The efficacy of radiation therapy (RT) in cervical cancer is well-established. Recently, however, cisplatin-based chemoradiation therapy (CRT) for locally advanced cervical cancer has shown a 30–50% survival benefit over RT alone (1). Despite this therapeutic innovation, pelvic recurrence was observed in 18–27% of patients even after CRT, and improvement of pelvic control is still needed (2, 3). Apoptosis, which means programmed cell death, is an active model of cell death that occurs in response to DNA damage by ionizing radiation, ultraviolet irradiation, and certain chemotherapeutic agents (4, 5). A large number of experimental studies have revealed that apoptosis induced by irradiation is a determining factor of radiosensitivity (6–9). Further, in RT for cervical cancer, a high occurrence of apoptotic cells after a total dose of 9 Gy was significantly associated with better pelvic control (10).
p73 belongs to the p53 tumor suppressor family of genes and shares significant homology with the p53 structural organization (11). Overexpression of p73 induced by irradiation activates the transcription of p53-responsive genes such as p21, Bax, Mdm2, and GADD45, and inhibits cell growth in a p53-like manner via the induction of apoptosis or cell-cycle arrest (12–15). In addition, p73 protein appears to be able to induce apoptosis independently of p53 in vitro (16, 17). However, it is unclear whether these mechanisms of apoptosis underlying p73 are also involved in the clinical effect of RT in cervical cancer (18). Many investigators have shown the impact of p53 status on treatment outcomes in patients with cervical cancer (6–8). Our previous study demonstrated that p53 mutation significantly worsened the local control of cervical cancer treated with RT (9). It is well known that E6 protein produced by human papillomavirus infection, which is associated with the development of cervical cancer, inactivates the normal
Reprint requests to: Tatsuya Ohno, MD, PhD, Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan. Tel: (+81) 43-206-3360; Fax: (+81) 43-256-6506; E-mail: [email protected] Supported by the National Institute of Radiological Sciences.
Conflict of interest: none. Acknowledgment—We thank M. Sakai for assisting with the TUNEL assay and immunohistochemistry. Received Nov 14, 2006, and in revised form May 30, 2007. Accepted for publication Aug 5, 2007.
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function of p53 (19). However, because the E6 protein does not inactivate p73 function, it would be interesting to know whether p73 can compensate for the impaired p53 function to trigger the apoptosis of cervical cancer cells in response to radiation (20). Here, we investigated the relation between p73 expression and radiation-induced apoptosis, with particular emphasis on whether p73 can compensate for impaired p53 function in the apoptosis induced by RT for cervical cancer. MATERIALS AND METHODS Patients Tumor samples obtained from 68 patients with cervical cancer who received RT alone or CRT at the National Institute of Radiological Sciences between 2002 and 2005 were investigated. All tumors were confirmed histologically as squamous cell carcinoma. Clinical stage was determined according to the classification of the International Federation of Gynecology and Obstetrics (21). The number of patients with Stage I, II, III, and IV disease was 5, 11, 37, and 15, respectively. Patient age ranged from 37 to 83 years, with a mean of 58 years. The study was approved by the Institutional Review Board of the National Institute of Radiological Sciences, and written informed consent was obtained from each patient before entry.
Treatment For RT, all patients were treated with a combination of external beam and high-dose-rate intracavitary irradiation. The standard treatment method for cervical cancer in our institute has been described previously (22). In brief, external irradiation to the whole pelvis was performed with anteroposterior and posteroanterior parallel opposed ports with a total dose of 30.6 Gy at 1.8 Gy per fraction, five times per week. This was followed by a central shielding pelvis field up to a total pelvis irradiation dose of 50.6 Gy at 2 Gy per fraction, five times per week. Along with the central shielding irradiation, intracavitary irradiation by a remote afterloading system using an iridium-192 source was performed. Four fractions of intracavitary irradiation were administered once per week at a fraction dose of 5–7 Gy at Point A, with the total dose ranging from 23 to 28 Gy. Among the 68 patients, 37 received CRT, which consisted of cisplatin at 40 mg/m2 weekly starting from week 1 for five consecutive weeks during the course of external beam irradiation. Eligibility criteria for CRT were as follows: Stage IIb >4 cm in diameter or Stage IIIb or IV disease; age between 20 and 70 years; performance status of 0–2; and adequate baseline bone marrow, hepatic, and renal function (white blood cell $3,000/mm3, hemoglobin $10 g/dL, platelet $100,000/mm3, total bilirubin #1.5 mg/dL, L-aspartate aminotransferase (AST)/L-alanine aminotransferase (ALT) #2 times the upper normal limit, and serum creatinine #1.5 mg/dL). Patients were excluded from CRT if they met any of the following criteria: prior chemotherapy or RT for the pelvis, severe pelvic infection, or severe psychological illness. All punch biopsy specimens were excised from the nonnecrotic cervical tumors before RT and after a total dose of 9 Gy (five fractions was given) for RT alone or 9 Gy plus one course of cisplatin for CRT. The second biopsy was taken from the same tumor area. All tissues were first fixed in 10% formalin for 1 day, embedded in paraffin, and sectioned into 3-mm serial sections. One section was stained with hematoxylin and eosin and the rest were subjected to the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method (TUNEL) assay for apoptotic cell defec-
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tion and immunohistochemical staining for p53 and p73 proteins. Tumor cell content was independently reviewed by a pathologist.
TUNEL assay Apoptotic cells were detected using the ApopTag Peroxidase in situ Apoptosis Detection kit (Chemicon International, Temecula, CA). The automated function of the Ventana Discovery System (Ventana Medical Systems, Tucson, AZ) was used from deparaffinization to counterstaining without titration of terminal deoxynucleotidyl transferase enzyme linkage of dUTP-digoxigenin to 30 -OH DNA ends. Briefly, formalin-fixed, paraffin-embedded sections (3 mm thick) from biopsy specimens were deparaffinized for 8 min by EZ Prep (Ventana Medical Systems). Protease 1 (Ventana Medical Systems) was treated for 30 min at 37 C. A mixture of ApopTag terminal deoxynucleotidyl transferase enzyme and ApopTag reaction buffer was applied for 40 min at 37 C, and then the reaction was stopped with ApopTag stop/wash buffer applied for 30 min at 37 C. Secondary antibody (monoclonal anti-DIGOXIN biotin conjugate; Sigma-Aldrich, St. Louis, MO) at a 1:500 dilution was applied for 52 min at 37 C. Finally, 3,3-diaminobenzidine (DAB) was used to detect the bound antibody complex. Slides were counterstained with hematoxylin. Apoptotic cells were identified on the basis of both TUNEL assay and characteristic morphological changes identified by hematoxylin and eosin staining, such as chromatin condensation and blebbing of the nucleus (Fig. 1). Artifact areas of nuclear staining associated with massive cellular necrosis were excluded from the analysis. The apoptotic cell index (AI) was calculated by dividing positive cells by the number of more than 2000 tumor cells in randomly selected fields in each case under a light microscope. In addition, AI ratio (AI after 9 Gy divided by AI before RT) was calculated to detect treatment-related apoptosis, and used to separate patients into high (AI ratio >2) and low AI ratio groups (AI ratio #2).
Immunohistochemistry Immunohistochemical studies were performed to detect the expressions of p53 and p73 proteins using a standard DAB-based immunostaining procedure employing a DAB Map Kit (Ventana Medical Systems). Sections were deparaffinized and cell
Fig. 1. Apoptotic cells stained by dUTP-biotin nick end labeling. Tumor cells with chromatin condensation and blebbing of the nucleus are positively stained, showing apoptotic cells (original magnification 400).
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Fig. 2. Immunohistochemical staining for p53 (A) and p73 (B) proteins. Immunostaining showed p53 (A) and p73 (B) positivity in the nucleus of tumor cells (original magnification 400). conditioning was performed (60 mins, 37 C) using the same automated Ventana system as above. Antibodies for p53 DO7 (Cell Marque, Hot Spring, AR) and p73 (Neomarkers, Fremont, CA) were diluted 100:1 and 50:1 in antibody dilution buffer (Ventana Medical Systems), respectively. Slides were incubated in the solution for 40 min. They were then incubated for 30 min with a universal secondary antibody (Ventana Medical Systems), counterstained with hematoxylin and postcounterstained with bluing reagent. Slides with high levels of p53 and p73 immunoreactivity were used as positive controls. Negative controls, without the primary antibody, were incubated in each step. Random sampling of more than 2,000 cells per tumor evaluated in three or four microscopic fields was then done. Positive staining for p53 or p73 protein was defined when more than 10% of tumor cells were stained with these proteins in each section (23, 24). In addition, in accordance with the staining response of p53 after 9 Gy of irradiation, samples were divided into a p53responding (p53 after 9 Gy/p53 before RT >1, and expression of p53 after 9 Gy >10%) and a p53–nonresponding group (p53 after 9 Gy/p53 before RT #1, or expression of p53 after 9 Gy #10%).
Statistical analysis Differences between AI before RT and AI after a total dose of 9 Gy were determined by means of the paired Student’s t-test. The unpaired Student’s t-test was used for the differences among AI, p53, and p73 positivity according to age group (>60 years vs. #60 years), stage (I and II vs. III and IV), and treatment (RT vs. CRT). The chisquare test was used for statistical analysis of the correlation between p53 expression or p73 expression and AI ratio. Fisher’s exact test was used for statistical analysis of the correlation between p73 expression after 9 Gy and AI ratio in the p53–nonresponding group. A p value of less than 0.05 was considered significant.
RESULTS Apoptotic index The mean AI was 0.93% (range, 0.02–3.06%) before irradiation and 1.97% (range, 0.26–4.83%) after 9 Gy, indicating a significant increase in AI in response to RT (p < 0.001). The high AI ratio group accounted for 40 (59%) of 68 patients and the low AI ratio group the other 28 (41%). Age (>60 years vs. #60 years), stage (I and II vs. III and IV), and combination of
chemotherapy were not associated with AI before RT or after 9 Gy. p53 and p73 expression p53 staining was seen in the nucleus of tumor cells (Fig. 2A). Thirty-four (50%) of 68 patients had positive p53 expression before RT and 44 (65%) were positive after 9 Gy, which was not a significant increase (p = 0.083). Based on our criteria, 41 (60%) patients were classified into the p53responding group and the remaining 27 (40%) into the p53– nonresponding group. Twenty-one (51%) of 41 patients received CRT in the p53-responding group, and 16 (59%) of 27 patients received CRT in the p53–nonresponding group. p73 staining was seen in the nucleus of tumor cells (Fig. 2B). Positive p73 expression was observed in 48 (71%) patients before RT and in 38 (56%) after 9 Gy. The difference in p73-positive rates before and after RT was not significant (p = 0.075), and no significant correlation between p53 and p73 expression before RT was seen (Table 1, p = 0.110). p53 expression positivity before RT was 19.5% for early stage (Stage I and II) and 10.2% for advanced stage (Stage III and IV) disease, with this difference being significant (p = 0.04). p73 expression positivity after 9 Gy was 9.4% for early stage (Stage I and II) and 16.9% for advanced stage (Stage III and IV) cancer, which was again significant (p = 0.03). Age (>60 years vs. #60 years) and combination
Table 1. Correlation between p53 and p73 expression before RT p53 before RT
p73 before RT
+ –
+
–
27 7
21 13
Abbreviation: RT = radiation therapy.
p = 0.110
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Table 2. Correlation between p53 or p73 expression before RT and AI ratio for all patients p53 before RT
AI ratio*
High(>2) Low(#2)
p73 before RT
+
–
20 14
20 14
p > 0.999
+
–
32 16
8 12
p = 0.042
Abbreviations: RT = radiation therapy; AI = apoptotic index. * Apoptotic index after 9 Gy/apoptotic index before RT.
of chemotherapy were not associated with p53 expression before RT and after 9 Gy, nor with p73 expression before RT and after 9 Gy. Correlation between apoptotic index and p53 or p73 expression As shown in Table 2, there was no significant correlation between p53 expression positivity before RT and AI ratio (p > 0.999). In contrast, a significant correlation between p73 expression positivity and AI ratio was observed before RT for all 68 patients (p = 0.042). As shown in Table 3, significant correlations were seen between p53 (p = 0.038) or p73 expression positivity (p = 0.021) after 9 Gy and AI ratio for all patients. In the p53-responding group, no significant correlation was seen between p73 expression after 9 Gy and AI ratio (p = 0.940, Table 4). In contrast, a significant positive correlation was seen between p73 expression after 9 Gy and AI ratio in the p53–nonresponding group (p < 0.001, Table 4). DISCUSSION In the present study, p73 expression positivity was 71% before RT. Singh et al. reported a positivity ratio before treatment of cervical cancer of 42% (19). Given that p73 positivity significantly increased with advancing disease stage in ovarian cancer, one reason for our higher positivity than that of Singh et al. may be differences in the proportion of patients with advanced disease (19, 25), with 76% of our patients having Stage III-IV disease vs. only 28% in their study. The current study showed no significant increase in p73 expression positivity after RT at 9 Gy, but did show a significant increase in that for p53. Several studies have reported irradiation-induced p73 expression in various tumor cells in vitro. Among them, Pucci et al. showed that p73 expression was induced by irradiation in glioblastoma cells lacking
the p53 gene (26). Lin et al. also showed that p73 levels were increased by irradiation in both p53 null mouse fibroblasts and p53 null human osteosarcoma cells (27). These studies suggested that p73 might be induced in cancer cells in which p53 function was absent. Further, several studies revealed that p73 was downregulated by p53 activation in breast cancer and lung cancer cells (28, 29). We therefore speculate that one reason that p73 expression did not significantly increase after 9 Gy was derived from a p53-induced negative feedback mechanism. The correlation of p73 expression with apoptosis in cervical cancer treated with RT remains unclear. Although p73 induces apoptosis by a different mechanism to p53 (30), it shares target genes with p53 (14, 31, 32). In the present study, no significant correlation was seen between p53 expression before RT and AI ratio, whereas p73 expression before RT correlated with AI ratio (p = 0.042). Further, a positive correlation was seen between p73 expression after 9 Gy and AI ratio, particularly in the p53–nonresponding group (p < 0.001). Irwin et al. reported that the induction of apoptosis was blocked by the inhibition of p73-dependent transcriptional activation in osteosarcoma cells, with a loss of p53 function (18). Schmid et al. also reported that doxorubicin increased p73 expression and induced the apoptosis of embryo fibroblast cells in a p53-lacking mouse (33). These experimental studies support our hypothesis that p73 may compensate for p53 function in the induction of apoptosis after irradiation for cervical cancer, when p53 function is inactivated by human papillomavirus infection. Prognosis has been shown to be poor in p73-positive patients undergoing surgery for colorectal adenocarcinoma, ovarian cancer and hepatocellular carcinoma (32, 34, 35). In contrast, p73 overexpression before RT was significantly associated with better survival in patients with cervical cancer (36). Given that it is possibly influenced by different treatment modalities and types of diseases, the prognostic
Table 3. Correlation between p53 or p73 expression after 9 Gy and AI ratio for all patients p53 after 9 Gy
AI ratio*
High(>2) Low(#2)
p73 after 9 Gy
+
–
31 15
9 13
Abbreviations as in Table 2. * Apoptotic index after 9 Gy/apoptotic index before RT.
p = 0.038
+
–
27 11
13 17
p = 0.021
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Table 4. Correlation between p73 expression after 9 Gy and AI ratio in the p53-responding group or the p53–nonresponding group p53-responding group p73 after 9 Gy
AI ratio*
High(>2) Low(#2)
+
–
16 8
12 5
p53–nonresponding group p73 after 9 Gy
p = 0.940
+
–
11 3
1 12
p < 0.001
Abbreviations as in Table 2. * Apoptotic index after 9 Gy/apoptotic index before RT.
value of p73 expression remains controversial. The prognostic significance of p73 expression in the present study could not be analyzed owing to the short follow-up period. To summarize, we confirmed the occurrence of radiationinduced apoptosis in patients with cervical cancer treated with RT or CRT by TUNEL assay and morphologic findings,
and of p53 and p73 expression by immunohistochemical methods. A significant positive correlation between p73 expression after 9 Gy and AI ratio was observed in the p53–nonresponding group. These results suggest that p73 may play a key role in the induction of apoptosis in cervical cancer patients with impaired p53 function.
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