[Inhibition of mutant-type p53 by a chimeric U6 maxizyme in hepatocellular carcinoma cell lines].
OBJECTIVE
To study the inhibition of maxizyme (Mz) directed against the mutant-type p53 gene (mtp53) at codon 249 in exon 7 (AGG --> AGT) both in cell-free system and in MHCC97 cell lines.


METHODS
Maxizyme and control mutant maxizyme (G5 --> A5) were designed by computer and cloned into the eukaryotic expression vector pBSKneoU6 (pU6Mz, pU6asMz). Mz was driven by T7 RNA polymerase promoter in vitro. In the cell lines, U6 promoter was driven by RNA PolIII. The mutant type p53 gene fragment was cloned into the pGEM-T vector under the T7 promoter control. The 32P-labeled mtp53 transcript was the target RNA. Cold maxizyme transcripts were incubated with 32P-labeled target RNA in vitro. pU6Mz was introduced into MHCC97 cells by Lipofectamine2000 and mtp53 expression was analyzed by RT-PCR and Western blot.


RESULTS
In vitro cleavage showed that pU6Mz was very active with cleavage efficiency of 42% while pU6asMz was not. The wild type p53 was not cleaved. Partial down-regulation of mtp53 mRNA and mtp53 protein were observed in MHCC97 cells transfected with pU6Mz but not those with pU6asMz. The proliferation of MHCC cells was inhibited by MTT analysis.


CONCLUSIONS
Our findings suggest that the chimeric U6 maxizyme against the mtp53 is a new promising gene therapeutic agent in treating hepatocellular carcinoma.
Kong
Xin-juan
XJ
Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Lin
Ju-sheng
JS
Song
Yu-hu
YH
Jin
You-xin
YX
D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D045744:Cell Line, Tumor; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics*; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D011487:Protein Conformation; D016337:RNA, Catalytic; D012333:RNA, Messenger / Q000138:chemical synthesis / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D006163:Ribonuclease T1 / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D016337:RNA, Catalytic; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; C427204:hammerhead ribozyme; D006163:Ribonuclease T1