RT-PCR amplification of RNA extracted from formalin-fixed, paraffin-embedded oral cancer sections: analysis of p53 pathway.
We present a new approach towards the detection of the mRNAs in formalin-fixed, paraffin-embedded samples using a reverse transcriptase (RT)-polymerase chain reaction (PCR). The total RNAs were extracted from 10-micron-thick sections and were reverse-transcribed, then the RT-products were subjected to PCR amplification of GAPDH mRNA for screening the mRNA degradation. Next, nested PCR was performed for examining the expression of p53-related genes, p21WAF1, MDM2, p33ING1 and p14ARF. GAPDH mRNA expression was detectable in 12 out of 21 oral squamous cell carcinoma (SCC) samples. p21WAF1 mRNA expression was detectable in 5 out of 12 SCC samples, MDM2 mRNA expression was detectable in 5 our of 12 SCC samples and p33ING1 mRNA expression was detectable in 6 out of 12 SCC samples. However, the expression of p14ARF mRNA was not detectable in any of the samples. Seven out of 12 oral SCC samples showed abnormal nuclear accumulation of p53 protein by immunohistochemical staining, whereas 5 out of 12 oral SCCs showed negative staining for p53 protein. Of of p33ING1 mRNA. One of these was a verrucous carcinoma in which the p53 gene products might be inactivated by the oncoprotein E6 of human papilloma virus. Thus, the p53 tumor suppressor pathway was disrupted in most oral SCCs at the cellular levels, due to either an abnormality in p53 itself or loss of expression of p53 regulatory factors. This method would assist in making diagnosis, determining therapeutic strategy and predicting the prognosis of various cancers including oral SCCs.
Tachibana
Masatsugu
M
Departments of Surgical and Molecular Pathology, Gastroenterological Surgery, Dokkyo University School of Medicine, 880 Kitakobayashi, Mibu, Shimo-tsuga, Tochigi, 321-0293, Japan.
Shinagawa
Yasuhiro
Y
Kawamata
Hitoshi
H
Omotehara
Fumie
F
Horiuchi
Hideki
H
Ohkura
Yasuo
Y
Kubota
Keiichi
K
Imai
Yutaka
Y
Fujibayashi
Takashi
T
Fujimori
Takahiro
T
D000818:Animals; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000096:biosynthesis / Q000235:genetics; D004268:DNA-Binding Proteins; D005557:Formaldehyde; D016147:Genes, Tumor Suppressor; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D000076023:Inhibitor of Growth Protein 1; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009062:Mouth Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D009687:Nuclear Proteins; D016612:Paraffin Embedding; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics; D051736:Proto-Oncogene Proteins c-mdm2; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016707:Tissue Fixation; D025803:Tumor Suppressor Protein p14ARF / Q000096:biosynthesis / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000235:genetics*; D025521:Tumor Suppressor Proteins
C493956:CDKN1A protein, human; C493957:Cdkn1a protein, mouse; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; C501503:ING1 protein, human; C501505:Ing1 protein, mouse; D000076023:Inhibitor of Growth Protein 1; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D025803:Tumor Suppressor Protein p14ARF; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D005557:Formaldehyde; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; C497206:MDM2 protein, human; C497207:Mdm2 protein, mouse; D051736:Proto-Oncogene Proteins c-mdm2