Maxizyme-mediated specific inhibition on mutant-type p53 in vitro.
OBJECTIVE
To evaluate the specific inhibition of maxizyme directing against mutant-type p53 gene (mtp53) at codon 249 in exon 7 (AGG-AGT) in vitro.


METHODS
Two different monomers of anti-mtp53 maxizyme (maxizyme right MzR, maxizyme left MzL) and control mutant maxizyme (G(5)-A(5)) were designed by computer and cloned into vector pBSKU6 (pBSKU6MzR, pBSKU6MzL). After being sequenced, the restrictive endonuclease site in pBSKU6MzR was changed by PCR and then U6MzR was inserted into pBSKU6MzL, the recombinant vector was named pU6Mz and pU6asMz (mutant maxizyme). Mtp53 and wild-type p53 (wtp53) gene fragments were cloned into pGEM-T vector under the T7 promoter control. The (32)p-labeled mtp53 transcript was the target mRNA. Cold maxizyme transcripts were incubated with (32)p-labeled target RNA in vitro and radioautographed after denaturing polyacrylamide gel electrophoresis.


RESULTS
In cell-free systems, pU6Mz showed a specific cleavage activity against target mRNA at 37 degrees and 25 mM MgCL(2). The cleavage efficiency of pU6Mz was 42 %, while pU6asMz had no inhibitory effect. Wtp53 was not cleaved by pU6Mz either.


CONCLUSIONS
pU6Mz had a specific catalytic activity against mtp53 in cell-free system. These lay a good foundation for studying the effects of anti-mtp53 maxizyme in HCC cell lines. The results suggest that maxizyme may be a promising alternative approach for treating hepatocellular carcinoma containing mtp53.
Kong
Xin-Juan
XJ
Institute of Liver Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China.
Song
Yu-Hu
YH
Lin
Ju-Sheng
JS
Huang
Huan-Jun
HJ
Wang
Nan-Xia
NX
Liu
Nan-Zhi
NZ
Li
Bin
B
Jin
You-Xin
YX
D006528:Carcinoma, Hepatocellular / Q000628:therapy; D002474:Cell-Free System; D003001:Cloning, Molecular; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D008113:Liver Neoplasms / Q000628:therapy; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D016337:RNA, Catalytic; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53