Relative quantitation of p53 and MDM2 gene expression in leiomyosarcoma; real-time semi-quantitative reverse transcription-polymerase chain reaction

To investigate the etiology of leiomyosarcoma, we examined abnormalities of p53 and its regulation in 13 cases of leiomyosarcoma using fresh tumor spe cimens. We estimated p53 and MDM2 mRNA level and MDM2 gene amplification us ing a real-time semi-quantitative reverse transcription-polymerase chain re action (RT-PCR) based on the TaqMan fluorescence method. We also used immun ohistochemistry (IHC) for p53 and MDM2 protein overexpression, polymerase c hain reaction single-strand conformation polymorphism (PCR-SSCP) and direct sequencing to detect p53 mutation. Eight of the 13 cases (62%) showed an o verexpression of p53 protein on IHC and eight of 13 cases (62%) had p53 gen e point mutations. Five of the 13 cases (38%) showed positive staining for MDM2 protein and only one case (7.7%) demonstrated MDM2 gene amplification. The relative p53 mRNA level of the tumors compared with normal tissue rang ed from 1.14 to 12.19 arbitrary units (AU), and the MDM2 mRNA level ranged from 1.06 to 17.17 AU. The mRNA level in the p53-positive cases was higher than in the negative cases (positive: 7.70 AU on average; negative: 3.38 AU on average; P = 0.0344). However, there was no significant correlation bet ween the MDM2 mRNA level and other factors, such as p53 IHC, p53 mutation s tatus, p53 mRNA level and MDM2 IHC. Our results indicate that p53 abnormali ties are major events and that an increasing level of p53 mRNA is associate d with an overexpression of p53 protein in leiomyosarcoma and they may play an important role in the tumorigenesis in this tumor. (C) 2001 Elsevier Sc ience Ireland Ltd. All rights reserved.