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. 2003 Jul;9(7):1571-5.
doi: 10.3748/wjg.v9.i7.1571.

Maxizyme-mediated specific inhibition on mutant-type p53 in vitro

Xin-Juan Kong  1 , Yu-Hu Song, Ju-Sheng Lin, Huan-Jun Huang, Nan-Xia Wang, Nan-Zhi Liu, Bin Li, You-Xin Jin
Affiliations

Maxizyme-mediated specific inhibition on mutant-type p53 in vitro

Xin-Juan Kong et al. World J Gastroenterol. 2003 Jul.

Abstract

Aim: 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.

Conclusion: 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.

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Figures

Figure 1
Figure 1
Structure of maxizyme against mtp53.
Figure 2
Figure 2
Steps of pU6Mz construction.
Figure 3
Figure 3
Restrictive enzyme analysis of recombinant plasmid pU6Mz by Sma I and Sac I. M was DNA Marker (DL2000). 1, 2, 3 were selected clones.
Figure 4
Figure 4
In vitro transcripts. (A) In vitro transcripts of wtp53 and mtp53 (1002nt). (B) In vitro transcripts of maxizyme and control maxizyme (910nt). lane 1: mtp53; lane 2: wtp53; lane 3: maxizyme; lane 4: control maxizyme.
Figure 5
Figure 5
In vitro cleavage reaction. lane 1: cleavage of wtp53 by cold maxizyme; lane 2: cleavage of mtp53 by inactive cold maxizyme; lane 3: cleavage of mtp53 by cold maxizyme.
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