Heidenreich A, Schenkman N S, Sesterhenn I A, Mostofi K F, Moul J W, Srivastava S, Engelmann U H
Department of Urology, University of Cologne, Germany.
APMIS. 1998 Jan;106(1):90-9; discussion 99-100. doi: 10.1111/j.1699-0463.1998.tb01324.x.
The role of p53 in testicular germ cell tumours is still contradictory based on the finding of immunohistochemical overexpression at the protein level, but lack of mutations at the DNA level. In addition, p53 wild-type activity has been demonstrated in cell culture experiments. Overexpression of the proto-oncogene bcl-2 might block p53-induced apoptosis and might inhibit p53 functional activity. To clarify the apparent paradox with respect to p53 overexpression and lack of mutations, an immunohistochemical and mutational analysis of p53 and bcl-2 in TGCT was performed. Ten normal testes, 52 CIS and 151 clinical stage I nonseminomatous GCTs were included in our study. A commercially available anti-p53 polyclonal rabbit antibody and an anti-bcl-2-mouse monoclonal antibody were used to stain the 5pm sections. Staining was assessed by counting at least 500 cells from the area of the most intense staining in each tumour cell type, and this was scored semiquantitatively for intensity of staining on a 4 point scale. In addition, 30 primary GCTs were included in the mutational analysis: areas with p53 overexpression were identified and microdissected prior to DNA extraction. p53 exons 5-8 were amplified by polymerase chain reaction (PCR) followed by single strand conformation polymorphism analysis. Templates demonstrating band shifts on SSCP were subjected to direct DNA sequence analysis. None of the normal testes, 32/52 (62%) CIS, and 142/151 (94%) germ cell tumours exhibited p53 overexpression. p53 expression was significantly lower in mature teratomas (0.8 +/- 0.2) than in other germ cell tumour components (2.8 +/- 1.2, p > 0.001). PCR-SSCP did not reveal any missense mutations or deletions for the p53 gene. Bcl-2 protein expression was observed in none of the normal testes, in none of the CIS, and in 14/151 (9.3%) germ cell tumours. 13/14 germ cell tumours demonstrated bcl-2 expression only in the glandular and stromal elements of their teratomatous components whereas all other components were negative for bcl-2. Our results--p53 overexpression, lack of p53 mutations, undetectable bcl-2--are consistent with recent in vitro studies. High susceptibility of testicular cancer to drug-induced apoptosis appears to be the result of wild-type p53 and lack of bcl-2. Radiation and chemotherapeutic insensitivity of mature teratomas might be the result of bcl-2 overexpression and lack of p53 overexpression. Therefore, chemoresistance to DNA damaging agents might be reflected by the expression of p53 and bcl-2 and it might be useful to evaluate p53 and bcl-2 in primary tumours and metastatic lesions in order to identify patients early with primary or secondary chemoresistance.
基于蛋白质水平免疫组化过表达但DNA水平缺乏突变这一发现，p53在睾丸生殖细胞肿瘤中的作用仍存在矛盾。此外，细胞培养实验已证明p53野生型活性。原癌基因bcl - 2的过表达可能会阻断p53诱导的凋亡，并可能抑制p53的功能活性。为了阐明p53过表达与缺乏突变这一明显的矛盾，我们对睾丸生殖细胞肿瘤中的p53和bcl - 2进行了免疫组化和突变分析。我们的研究纳入了10个正常睾丸、52个原位癌（CIS）和151个临床I期非精原细胞瘤性生殖细胞肿瘤（GCT）。使用市售的抗p53多克隆兔抗体和抗bcl - 2小鼠单克隆抗体对5微米切片进行染色。通过对每种肿瘤细胞类型中染色最强烈区域的至少500个细胞进行计数来评估染色情况，并根据染色强度在4分制上进行半定量评分。此外，30个原发性GCT纳入突变分析：在DNA提取之前，先识别并显微切割p53过表达区域。通过聚合酶链反应（PCR）扩增p53外显子5 - 8，随后进行单链构象多态性分析。对在单链构象多态性分析（SSCP）中显示条带迁移的模板进行直接DNA序列分析。正常睾丸、32/52（62%）的原位癌和142/151（94%）的生殖细胞肿瘤均未表现出p53过表达。成熟畸胎瘤中的p53表达（0.8±0.2）显著低于其他生殖细胞肿瘤成分（2.8±1.2，p>0.001）。PCR - SSCP未发现p53基因的任何错义突变或缺失。在正常睾丸、原位癌中均未观察到bcl - 2蛋白表达，在14/151（9.3%）的生殖细胞肿瘤中观察到bcl - 2蛋白表达。13/14的生殖细胞肿瘤仅在其畸胎瘤成分的腺上皮和基质成分中显示bcl - 2表达，而所有其他成分bcl - 2均为阴性。我们的结果——p53过表达、p53无突变、未检测到bcl - 2——与最近的体外研究一致。睾丸癌对药物诱导凋亡的高敏感性似乎是野生型p53和缺乏bcl - 2的结果。成熟畸胎瘤对放疗和化疗不敏感可能是bcl - 2过表达和缺乏p53过表达的结果。因此，对DNA损伤剂的化疗耐药性可能通过p53和bcl - 2的表达反映出来，评估原发性肿瘤和转移灶中的p53和bcl - 2对于早期识别原发性或继发性化疗耐药的患者可能是有用的。
