Tuberc Respir Dis TRD Tuberculosis and Respiratory Diseases 1738-3536 The Korean Academy of Tuberculosis and Respiratory Diseases 10.4046/trd.2007.63.3.251 Original Article Expression of Caspase 3, Survivin, and p53 Protein in Urethane Induced Mouse Lung Carcinogenesis Shin Jong Wook M.D. 1 Lee Soo Hwan M.D. 2 Park Eon Sub M.D. 2 Division of Allergy, Respiratory and Critical Care Medicine, Department of Internal Medicine, Chung Ang University College of Medicine, Seoul, Korea. Department of Pathology, Chung Ang University College of Medicine, Seoul, Korea. Address for correspondence: Jong Wook Shin, M.D. and Eon Sub Park, M.D. Division of Allergy, Respiratory and Critical Care Medicine, Department of Internal Medicine, Department of Pathology, Chung-Ang University Hospital, 224-1, Huk-suk-dong, Donjak-gu, Seoul, Korea. Phone: 82-2-6299-1407, Fax: 82-2-6263-2184, basthma@cau.ac.kr, basthma@hanmail.net, esp@cau.ac.kr 09 2007 30 09 2007 63 3 251 260 03 05 2007 14 09 2007 Copyright © 2007 The Korean Academy of Tuberculosis and Respiratory Diseases 2007 Purpose

An imbalance of cell proliferation and cell apoptosis is an important mechanism in carcinogenesis. Capase 3, survivin and p53 have been identified as important members of the apoptotic related proteins. This study evaluated the proliferating cell nuclear antigen(PCNA), apoptosis, apoptotic related protein such as capase 3, survivin and p53 using urethane-induced mouse lung carcinogenesis, which provides reproducible steps from hyperplasia to adenocarcinoma.

 Methods

Urethane was administered to the ICR mice through an intra-peritoneal injection, The mice were sacrificed at 5, 15, and 25 weeks after urethane intervention. The sequential morphological changes and immunohistochemical expression of PCNA, apoptosis, capase 3, survivin, and p53 were examined during mouse lung carcinogenesis.

 Results

During carcinogenesis, the sequential histological changes were observed from hyperplasia of type II pneumocytes, to anadenoma, and ultimately to an overt adenocarcinoma. The PCNA Labeling index (LI) was 9.6% in hyperplasia, 23.2% in adenoma, and 55.7% in adenocarcinoma, respectively. The apoptotic LI was 0.24% in hyperplasia, 1.25% in adenoma, and 5.27% in adenocarcinoma. A good correlation was observed between the PCNA LI and apoptotic LI. The expression of caspase 3 was remarkable- i.e., 46.7% in adenocarcinoma, in contrast to 15% in hyperplasia and 16% in adenoma. Survivin was detected weakly in the alveolar hyperplasia and showed an increasing expressional pattern in adenoma and adenocarcinoma. p53 expression was detected only in the adenocarcinoma lesions with an expression rate of 13.3%. The level of caspase 3 expression correlated with the increase in the apoptotic index. The positive expression of caspase 3 was associated with an increased apoptotic index.

 Conclusions

These results suggest that the PCNA LI and apoptotic LI might be useful markers for evaluating the risk of a malignant transformation. In addition, caspase, survivin and p53 might play a role in the early and late steges of urethane-induced mouse lung carcinogenesis.

 Survivin caspase-3 p53 Lung cancer Lung carcinogenesis PCNA Apoptosis Zang EA Wynder AL Cumulative tar exposure. A new index for estimating lung cancer risk among cigarette smokers Cancer 1992 70 69 76 Foley JF Anderson MW Stoner GD Gaul BW Hardisty JF Maronpot RR Proliferative lesions of the mouse lung: progression studies in strain A mice Exp Lung Res 1991 17 157 168 Malkinson AM Pulmonary lung tumors in mice: an experimentally manipulable model of human adenocarcinoma Cancer Res 1992 52 2670s 2676s Re FC Manenti G Borrello MG Colombo MP Fisher JH Pierotti MA Multiple molecular alterations in mouse lung tumors Mol Carcinog 1992 5 155 160 Hartwell LH Kastan MB Cell cycle control and cancer Science 1994 266 1821 1828 Robbins BA Vega D Ogata K Tan EM Nakamura RM Immunohistochemical detection of proliferating cell nuclear antigen in solid human malignancies Arch Pathol Lab Med 1987 111 841 845 Cox LS Multiple pathways control cell growth and transformation: overlapping and independent activities of p53 and p21 J Pathol 1997 183 134 140 Grossman D McNiff JM Li F Altieri DC Expression and targeting of the apoptosis inhibitor, survivin, in human melanoma J Invest Dermatol 1999 113 1076 1081 Gavrieli Y Sherman Y Ben-Sasson SA Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation J Cell Biol 1992 119 493 501 Altieri DC Marchisio C Survivin apoptosis: an interloper between cell death and cell proliferation in cancer Lab Invest 1999 79 1327 1333 Alnemri ES Livinston DJ Nicholson DW Salvesen G Thornberry NA Wong WW Human ICE/CED-3 protease nomenclature Cell 1996 87 171 Mirza A McGuirk M Hockenberry TN Wu Q Ashar H Black S Human survivin is negatively regulated by wild-type p53 and participates in p53 dependent apoptotic pathway Oncogene 2002 21 2613 2622 Chung JH Jang JJ Lee MJ Ham EK Altered retinoblastoma protein expression and proliferative activity in urethane induced mouse lung tumorigenesis Cancer Res Treat 2002 34 258 263 Bravo R Frank R Blundell PA Macdonald-Bravo H Cyclin/PCNA is the auxillary proteins of DNA polymerase-delta Nature 1987 326 515 517 Morris GF Mathews MB Regulation of proliferating cell nuclear antigen during the cell cycle J Biol Chem 1989 264 13856 13864 Kelman Z PCNA: structure, functions and interactions Oncogene 1997 14 629 640 Tormanen U Eerola AK Rainio P Vahakangas K Soini Y Sormunen R Enhanced apoptosis predicts shortened survival in non-small cell lung carcinoma Cancer Res 1995 55 5595 5602 Lipponen PK Aaltomaa S Apoptosis in bladder cancer as related to standard prognostic factors and prognosis J Pathol 1994 173 333 339 Shibata MA Maroulakou IG Jorcyk CL Gold LG Ward JM Green JE p53 independent apoptosis during mammary tumor progression in C(3)/SV 40 large T antigen transgenic mice; Supression of apoptosis during the transition from preneoplasia to carcinoma Cancer Res 1996 56 2998 3003 Sela B Survivin: anti-apoptosis proteins and a prognostic markers for progression and recurrence Harefuah 2002 141 103 107 Ito T Shiraki K Sugimoto K Yamanaka T Fujikawa K Ito M Survivin promotors cell proliferation in human hepatocellular carcinoma Hepatology 2000 31 1080 1085 Lin LJ Zheng CQ Jin Y Ma Y Jiang WG Ma T Expression of survivin protein in human colorectal carcinogenesis World J Gastroenterol 2003 9 974 977 Zhang X Dong N Yin L Cai N Ma H You J Hepatitis B virus X protein upregulates survivin expression in hepatoma tissues J Med Virol 2005 77 374 381 Akyurek N Memis L Ekinci O Kokturk N Ozturk C Survivin expression in pre-invasive lesions and non-small cell lung carcinoma Virchows Arch 2006 449 164 170 Hsia JY Chen CY Chen JT Hsu CP Shai SE Yang SS Prognostic significance of caspase-3 expression in primary resected esophageal squamous cell carcinoma Eur J Surg Oncol 2003 29 44 48 Li YH Wang C Meng K Chen LB Zhou XJ Influence of survivin and caspase-3 on cell apoptosis and prognosis in gastric carcinoma World J Gastroenterol 2004 10 1984 1988 Isobe N Onodera H Mori A Shimada Y Yang W Yasuda S Caspase-3 expression in human gastric carcinoma and its clinical significance Oncology 2004 66 201 209 Hall PA Lane DP p53 in tumor pathology: can we trust immunohistochemistry? J Pathol 1994 172 1 4 Cazorla M Hernandez L Fernandez PL Fabra A Peinado MA Dasenbrock C Ki-ras gene mutations and absence of p53 gene mutations in spontaneous and urethane-induced early lung lesions in CBA/J mice Mol Carcinog 1998 21 251 260 Horio Y Chen A Rice P Roth JA Malkinson AM Schrump DS Ki-ras and p53 mutations are early and late events, respectively, in urethane-induced pulmonary carcinogenesis in A/J mice Mol Carcinog 1996 17 217 223 Ohno J Horio Y Sekido Y Hasegawa Y Takahashi M Nishizawa J Telomerase activation and p53 mutations in urethane-induced A/J mouse lung tumor development Carcinogenesis 2001 22 751 756 Mori I Yasuhara K Hayashi SM Nonoyama T Nomura T Yanai T Aberrant expression of cyclin D1 in pulmonary proliferative lesions induced by high doses of urethane in transgenic mice carrying the human prototype c-H-ras gene J Vet Med Sci 2001 63 261 268

A. Gross finding of urethane-induced lungs, which shows multiple masses. B, C. Sequential change in urethane-induced lung lesions, showing adenoma (Figure 1B) and infiltrating adenocarcinoma (Figure 1C)(Hematoxylline & Eosin Stain, ×200). D. Immunohistochemical reactivity for PCNA in adenoma (×200). E. TUNNEL stain shows occasional nuclear staining in adenocarcinoma of lung (×200). F, G, H. Immunohistochemical reactivity for capase 3 (Figure 1F), p53 (Figure 1G) and survivin protein (Figure 1H) showing positive cytoplasmic staining (×200).

Histologic changes of the lung in mice treated with urethane

Tumor multiplicity; Number of tumor per mouse.

PCNA and apoptotic labeling index of the mice lung lesions during urethane-induced carcinogenesis

Significantly greater than the other groups, p<0.01.

Expression rates of caspase-3, survivin and p53 protein in mouse lung lesions by urethane-induced lung carcinogenesis

NS: not significantly different.

Proliferation and apoptotic index according to caspase 3 and survivin expressin expression

n: number of tumor lesion; NS: not significantly different. Significantly greater than the other groups, p<0.01.