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 specimens. We estimated p53 and MDM2 mRNA level and MDM2 gene amplification using a real-time semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) based on the TaqMan fluorescence method. We also used immunohistochemistry (IHC) for p53 and MDM2 protein overexpression, polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and direct sequencing to detect p53 mutation. Eight of the 13 cases (62%) showed an overexpression of p53 protein on IHC and eight of 13 cases (62%) had p53 gene 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 ranged 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 between the MDM2 mRNA level and other factors, such as p53 IHC, p53 mutation status, p53 mRNA level and MDM2 IHC. Our results indicate that p53 abnormalities are major events and that an increasing level of p53 mRNA is associated with an overexpression of p53 protein in leiomyosarcoma and they may play an important role in the tumorigenesis in this tumor.
Miyajima
K
K
Department of Anatomic Pathology, (Second Department of Pathology), Pathological Sciences, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, 812-8582, Fukuoka, Japan.
Tamiya
S
S
Oda
Y
Y
Adachi
T
T
Konomoto
T
T
Toyoshiba
H
H
Masuda
K
K
Tsuneyoshi
M
M
D000328:Adult; D000368:Aged; D004252:DNA Mutational Analysis; D005091:Exons; D005260:Female; D016158:Genes, p53 / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D007890:Leiomyosarcoma / Q000235:genetics* / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D009154:Mutation; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis*; D051736:Proto-Oncogene Proteins c-mdm2; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis*
D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2