KISEP Head and Neck Korean J Otolaryngol 2000;43:523-7 구강편평세포암에서 p53, p21, Bax 단백의면역조직화학적발현양상및예후적의의 김상현 황동조 김정수 문준환 노호상 The Expression Pattern and the Prognostic Value of p53, p21, and Bax Proteins in Oral Squamous Cell Cancer Sang-Hyun Kim, MD, Dong-Jo Hwang, MD, Jeong-Soo Kim, MD, Jun-Hwan Moon, MD and Ho-Sang No, MD Department of Otolaryngolgy, National Medical Center, Seoul, Korea ABSTRACT Background and ObjectivesThe wild-type p53 protein activates different tumor suppressor genes, leading to the G1 arrest following DNA damage. P21 and bax play a pivotal role in the regulation of apoptosis. The authors wanted to find out the relationship among p53, p21 and bax and the correlation between the staining results and clinicopathologic factors. We also assessed their influence on survival in the oral cavity cancer. Materials and MethodsParaffin embedded tissue sections were made from squamous cell carcinoma of oral cavity from 46 patients. Tissue sections were immunohistochemically stained for the expression of p53, p21 and bax. Results were then compared with the size of primary tumor, lymph node metastasis and histopathologic differentiation. The probability of survival was calculated by the Kaplan-Meier method. ResultsImmunoreactivity of the p53 and p21 were detected dominantly in the nuclei at various levels, and immunoreativity of bax was detected in the cytoplasm. The positive rates of p53, p21, and bax were 54.4%, 58.7%, 26.1%, respectively. The positive rate of p53 and the negative rate of bax expression were significantly increased with T-stage but were not affected by N-stage and histopathologic differentiation. p21 had no correlation with the T-stage, N-stage nor with pathologic differentiation. In the multivariate analysis, neither the single oncoprotein nor the combinations of p53, p21, bax had influence on survival statistically. ConclusionThe bax protein and mutant p53 protein can be a biological marker for primary tumor progression. Korean J Otolaryngol 2000;43:523-7 KEY WORDSSquamous cell carcinoma Protein p53 p21waf1 Bax Prognosis. 523
구강암에서 p53, p21, Bax 단백의 발현양상 및 예후와의 연관성 요구되고 있다.5) 이미 Apolinario 등1)과 Lee 등2)은 면역 (Calbio, England)를 1 20으로, bax에 대한 일차항체로 조직화학적으로 단백발현된 양상과 생존률과의 비교연구를 는 rabbit polyclonal antibodies인 bax(ab-1). (Calbio, 통하여 각 유전자의 발현이 갖는 예후인자로써의 가치에 England)를 1 20으로 희석하여 각각 37 에서 2시간동 대해 논제를 제기한 바 있다. 이에 저자들은 편평세포암으 로 진단된 구강암 환자의 파라핀포매조직을 이용하여, 면역 조직화학적 연구방법을 통해 각 종양억제유전자의 발현양 상이 갖는 상호연관성과 생존률에 대한 비교분석 및 원발 병소의 병기, 경부림프절전이, 병리조직학적 분화도 등과의 관련성에 대한 통계적 분석을 통하여 종양억제유전자의 발 현양상이 갖는 예후인자로써의 의의를 알아보고자 하였다. 재료 및 방법 연구대상 및 재료 1980년 1월부터 1997년 12월까지 본원에서 구강 편평 세포암의 진단 하에 수술 치료를 시행한 환자 중 방사선치 료나 화학약물치료에 전혀 노출된 기왕력이 없으며 파라핀 Fig. 1. Strong p53-positive nuclear staining (arrows) was seen in the tongue cancer cell (Immunohistochemical staining, 400). 포매 조직의 이용이 가능한 46명의 환자를 대상으로 하였 다. 이중 병변에 대한 의무기록이 충분치 못한 6례는 각 단 백 상호간의 분석시에만 포함하였고 임상적, 병리적 소견 과의 상관관계 분석시에는 제외하였다. 환자의 평균나이는 56.7세로 31세부터 80세까지 다양했으며 남녀의 성비는 6 1(39 7)로 남자에서 빈발하였고 발생부위별 빈도는 설암이 24례로 가장 많았으며, 치주 8례, 경구개 8례, 구강 저부 3례, 후구삼각부 2례, 구강점막 1례 순 이었다. 면역조직화학적 염색방법 수술시 적출되어 파라핀 포매되어 보관중인 조직 중 종양 세포가 가장 광범위하게 포함된 것을 골라 5 μm 두께의 연속 절편으로 만들어 silanized slide(dako corp.)에 부 Fig. 2. Strong p21-positive nuclear staining (arrows) was seen in the tongue cancer cell (Immunohistochemical staining, 400). 착시켰고, Xylene에 각각 10분간씩 3회 담가 탈파라핀화 과정을 거친 후 100%, 90%, 80%, 70% 에탄올에 3분간 씩 처리하여 함수과정을 거쳤으며 내인성과산화효소의 활성 을 억제시키기 위하여 메탄올-과산화수소용액(100% methanol 160 ml+30% H2O2 4 cc)에 10분간 처리하였다. ph 7.6의 Tris 완충액으로 5분간씩 3회 세척하였고 각각 의 슬라이드는 ph 6.0 citrate buffer 용액에 충분히 잠기 게 하여 microwave oven에서 15분간 가열하였다. 상온에 서 PBS 용액에 20분간 방치하여 냉각한 후 각 단백에 대 한 일차항체를 반응시켰는데 p53에 대한 일차항체는 mouse monoclonal antibody인 clone 1801(BioGenex, USA)를 1 50으로 희석하여 사용하였고, p21에 대한 일차항체는 mouse monoclonal antibody인 clone EA10WAF1(Ab-1). 524 Fig. 3. Strong bax-positive cytoplasmic staining (arrows) was seen in the tongue cancer cell (Immunohistochemical staining, 400). Korean J Otolaryngol 2000;43:523-7
Table 1. Expression of p53, p21, bax Table 3. Correlation between the expression of p53, p21, bax and clinicopathologic factors T stage p53 % p21 % bax % Positive 25 54.4 27 58.7 12 26.1 Negative 21 45.6 19 41.3 34 73.9 Total 46 100 46 100 46 100 Table 2. Correlation between the expression of p53 and p21 and bax p21 p21 bax bax Total p53 12 13 4 21 25 p53 15 6 8 13 21 Total 27 19 12 34 46 p0.140 p0.107 Fig. 4. Cumulative survival Kaplan-Meier analysis curves for patients with oral cavity cancer. p53 p21 Bax T1 8 1 4 5 3 6 9 T2 7 7 5 9 10 4 14 T3 5 9 6 8 13 1 14 T4 0 3 3 0 3 0 3 p0.016 p0.309 p0.017 N stage No 12 11 10 13 16 7 23 N13 8 9 8 9 13 4 17 p0.819 p0.745 p0.787 Pathologic differentiation Poor 3 2 4 1 3 2 5 Moderate 3 6 4 5 9 0 9 Well 14 12 10 16 17 9 26 p0.633 p0.263 p0.087 Total 525
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