Gumerlock P H, Chi S G, Shi X B, Voeller H J, Jacobson J W, Gelmann E P, deVere White R W
Department of Internal Medicine, University of California, Davis Cancer Center, Sacramento 95817, USA.
J Natl Cancer Inst. 1997 Jan 1;89(1):66-71. doi: 10.1093/jnci/89.1.66.
The reported frequency of mutation of the p53 tumor suppressor gene (also known as TP53) in human carcinomas of the prostate has varied widely, ranging from 3% to 42%. This variability may be a consequence of tumor heterogeneity and/or the use of different methods of analysis. Since p53 mutation has been associated with clinical outcome for a number of cancer types, determination of its true frequency in primary carcinomas of the prostate is important.
The principal aims of this study were as follows: 1) to validate the utility of detecting p53 gene mutations by means of polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis of complementary DNA (cDNA) (synthesized from prostate tissue RNA and 2) to study the concordance of RNA- and DNA-based PCR-SSCP assays in detecting p53 mutations in individual tumor fragments.
RNA and genomic DNA were isolated by means of standard techniques from specimens of 19 carcinomas of the prostate, selected on the basis of p53 data obtained in a previous analysis of cDNA (indicating that 14 were mutant and five were wild-type). RNA was converted into cDNA by means of reverse transcription (RT); the cDNA was then amplified by means of nonisotopic (i.e., nonradioactive) PCR, and the PCR products were subjected to SSCP analysis in polyacrylamide gels (RT-PCR-SSCP analysis). Genomic DNA was examined by means of SSCP analysis of isotopically labeled (32PO4) PCR products (DNA-PCR-SSCP analysis). In both approaches, the protein coding region of the p53 gene was divided into multiple, smaller fragments for study. PCR products exhibiting abnormal migration in SSCP gels were subjected to direct nucleotide sequencing or to cloning and sequencing of multiple clones.
RT-PCR-SSCP and DNA-PCR-SSCP identified p53 gene abnormalities in 15 of the 19 selected carcinomas, including one previously reported to be wild-type for p53. Overall, PCR-SSCP analysis identified 18 p53 fragments with abnormalities; three carcinomas showed two abnormalities each. Six (33%) of the 18 abnormalities were detected by both RT-PCR-SSCP and DNA-PCR-SSCP, 10 (56%) were detected by RT-PCR-SSCP alone, and two (11%) were detected by DNA-PCR-SSCP alone. The 18 abnormalities were caused by 20 changes in the sequence of the p53 gene; in one carcinoma, double mutations in two individual p53 exons were identified.
PCR-SSCP analysis of both RNA and DNA allows the detection of more mutations than the analysis of either alone. Some primary carcinomas of the prostate contain more than one altered p53 gene, consistent with the possibility of intratumoral heterogeneity of mutation of this gene. For comprehensive analysis of p53 mutations in carcinomas of the prostate, and perhaps in other tumor tissues, SSCP analysis of cDNA should be used in combination with SSCP analysis of genomic DNA.
据报道，p53肿瘤抑制基因（也称为TP53）在人类前列腺癌中的突变频率差异很大，从3%到42%不等。这种变异性可能是肿瘤异质性和/或使用不同分析方法的结果。由于p53突变已与多种癌症类型的临床结果相关，因此确定其在前列腺原发性癌中的真实频率很重要。
本研究的主要目的如下：1）通过对互补DNA（cDNA）（由前列腺组织RNA合成）进行聚合酶链反应-单链构象多态性（PCR-SSCP）分析来验证检测p53基因突变的实用性，以及2）研究基于RNA和DNA的PCR-SSCP检测在单个肿瘤片段中检测p53突变的一致性。
通过标准技术从19例前列腺癌标本中分离RNA和基因组DNA，这些标本是根据先前对cDNA的分析获得的p53数据（表明14例为突变型，5例为野生型）选择的。RNA通过逆转录（RT）转化为cDNA；然后通过非同位素（即非放射性）PCR扩增cDNA，并将PCR产物在聚丙烯酰胺凝胶中进行SSCP分析（RT-PCR-SSCP分析）。通过对同位素标记（32PO4）的PCR产物进行SSCP分析来检测基因组DNA（DNA-PCR-SSCP分析）。在这两种方法中，p53基因的蛋白质编码区域被分成多个较小的片段进行研究。在SSCP凝胶中表现出异常迁移的PCR产物进行直接核苷酸测序或对多个克隆进行克隆和测序。
RT-PCR-SSCP和DNA-PCR-SSCP在19例所选前列腺癌中的15例中鉴定出p53基因异常，其中包括1例先前报道为p53野生型的病例。总体而言，PCR-SSCP分析鉴定出18个p53片段存在异常；3例癌症各显示出2个异常。18个异常中有6个（33%）通过RT-PCR-SSCP和DNA-PCR-SSCP均检测到，10个（56%）仅通过RT-PCR-SSCP检测到，2个（11%）仅通过DNA-PCR-SSCP检测到。这18个异常是由p53基因序列中的20个变化引起的；在1例癌症中，在两个单独的p53外显子中鉴定出双突变。
对RNA和DNA进行PCR-SSCP分析比单独分析其中任何一种能检测到更多的突变。一些前列腺原发性癌含有不止一个改变的p53基因，这与该基因肿瘤内突变异质性的可能性一致。为了对前列腺癌以及可能其他肿瘤组织中的p53突变进行全面分析，应将cDNA的SSCP分析与基因组DNA的SSCP分析结合使用。
