Construction of Retroviral Vector of P(125FAK) Specific Ribozyme Genes and Its Effects on BGC-823 Cells

Overview

Journal World J Gastroenterol

Publisher Baishideng Publishing Group

Specialty Gastroenterology

Date 2006 Mar 8

PMID 16521179

Citations 1

Authors

Guo-Xian Guan

Hong-Xing Jian

Dong-Yin Lei

Hui-Shan Lu

Xiang-Fu Zhang

Affiliations

Soon will be listed here.

Abstract

Aim: To construct the retroviral vector of p(125FAK) specific ribozyme genes and to explore the feasibility of ribozyme in BGC-823 gene therapy in vitro.

Methods: A hammerhead ribozyme DNA targeting p(125FAK) mRNA from nt 1010 to nt 1032 was synthesized and recombined into the retroviral vector pLXSN forming pLRZXSN recon. Using the lipofectin-mediated DNA transfection technique, pLRZXSN was introduced into BGC-823 cells. The effects of ribozyme on the growth of BGC-823 cells and apoptosis were studied by cell colony assay, flow cytometry (FCM), reverse transcriptase-polymerase chain reaction (RT-PCR), detection of DNA fragmentation and electron microscopy.

Results: The number of BGC-823 cell colonies was inhibited by 56% after the cells were treated for 48 h. The cell proliferation was inhibited effectively by p(125FAK) ribozyme and the inhibitory effect depended on the concentration and the time of incubation. The expression of p(125FAK) mRNA and protein P(125FAK) decreased sharply in BGC-823 cells treated with p(125FAK) ribozyme. The characteristics of apoptosis, namely sub-G1 peak, DNA fragmentation and morphological changes, were revealed in BGC-823 cells treated with p(125FAK) ribozyme.

Conclusion: p(125FAK) ribozyme decreases p(125FAK) gene expression and induces apoptosis of human gastric cancer cells in vitro.

Citing Articles

Expression and clinical significance of focal adhesion kinase in the two distinct histological types, intestinal and diffuse, of human gastric adenocarcinoma.

Giaginis C, Vgenopoulou S, Tsourouflis G, Politi E, Kouraklis G, Theocharis S Pathol Oncol Res. 2008; 15(2):173-81.

PMID: 18987997 DOI: 10.1007/s12253-008-9120-2.

References

Hauck C, Sieg D, Hsia D, Loftus J, Gaarde W, Monia B . Inhibition of focal adhesion kinase expression or activity disrupts epidermal growth factor-stimulated signaling promoting the migration of invasive human carcinoma cells. Cancer Res. 2001; 61(19):7079-90. View

Song Y, Chen X, Kong X, Liu N, Li W, Wu X . Ribozymes against TGFbeta1 reverse character of activated hepatic stellate cells in vitro and inhibit liver fibrosis in rats. J Gene Med. 2005; 7(7):965-76. DOI: 10.1002/jgm.744. View

Kasahara T, Koguchi E, Funakoshi M, Aizu-Yokota E, Sonoda Y . Antiapoptotic action of focal adhesion kinase (FAK) against ionizing radiation. Antioxid Redox Signal. 2002; 4(3):491-9. DOI: 10.1089/15230860260196290. View

Beviglia L, Golubovskaya V, Xu L, Yang X, Craven R, Cance W . Focal adhesion kinase N-terminus in breast carcinoma cells induces rounding, detachment and apoptosis. Biochem J. 2003; 373(Pt 1):201-10. PMC: 1223465. DOI: 10.1042/BJ20021846. View

Golubovskaya V, Gross S, Kaur A, Wilson R, Xu L, Yang X . Simultaneous inhibition of focal adhesion kinase and SRC enhances detachment and apoptosis in colon cancer cell lines. Mol Cancer Res. 2003; 1(10):755-64. View

Gu Y, Chen J, Zhou X . [Inhibitory effects of antisense focal adhesion kinase oligodeoxynucleotides on the invasion of Bel 7402 hepatocellular carcinoma cells]. Zhonghua Gan Zang Bing Za Zhi. 2003; 11(10):612-5. View

Kijima H, Yamazaki H, Nakamura M, Scanlon K, Yoshiyuki Osamura R, Ueyama Y . Ribozyme against mutant K-ras mRNA suppresses tumor growth of pancreatic cancer. Int J Oncol. 2004; 24(3):559-64. View

Shin K, Sullenger B, Lee S . Ribozyme-mediated induction of apoptosis in human cancer cells by targeted repair of mutant p53 RNA. Mol Ther. 2004; 10(2):365-72. DOI: 10.1016/j.ymthe.2004.05.007. View

Abuharbeid S, Apel J, Sander M, Fiedler B, Langer M, Zuzarte M . Cytotoxicity of the novel anti-cancer drug rViscumin depends on HER-2 levels in SKOV-3 cells. Biochem Biophys Res Commun. 2004; 321(2):403-12. DOI: 10.1016/j.bbrc.2004.06.160. View

10.

Zhang Y, Taylor M, Samson W, Phillips M . Antisense inhibition: oligonucleotides, ribozymes, and siRNAs. Methods Mol Med. 2004; 106:11-34. View

11.

Haseloff J, Gerlach W . Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature. 1988; 334(6183):585-91. DOI: 10.1038/334585a0. View

12.

Symons R . Small catalytic RNAs. Annu Rev Biochem. 1992; 61:641-71. DOI: 10.1146/annurev.bi.61.070192.003233. View

13.

Juliano R, Haskill S . Signal transduction from the extracellular matrix. J Cell Biol. 1993; 120(3):577-85. PMC: 2119550. DOI: 10.1083/jcb.120.3.577. View

14.

Chen Y, Gu X, Caen J, Han Z . Interleukin-3 is an autocrine growth factor of human megakaryoblasts, the DAMI and MEG-01 cells. Br J Haematol. 1994; 88(3):481-7. DOI: 10.1111/j.1365-2141.1994.tb05063.x. View

15.

Xu L, Owens L, Sturge G, Yang X, Liu E, Craven R . Attenuation of the expression of the focal adhesion kinase induces apoptosis in tumor cells. Cell Growth Differ. 1996; 7(4):413-8. View

16.

Xu L, Yang X, Craven R, Cance W . The COOH-terminal domain of the focal adhesion kinase induces loss of adhesion and cell death in human tumor cells. Cell Growth Differ. 1998; 9(12):999-1005. View

17.

Tamura M, Gu J, Takino T, Yamada K . Tumor suppressor PTEN inhibition of cell invasion, migration, and growth: differential involvement of focal adhesion kinase and p130Cas. Cancer Res. 1999; 59(2):442-9. View

18.

Scanlon K . Anti-genes: siRNA, ribozymes and antisense. Curr Pharm Biotechnol. 2004; 5(5):415-20. DOI: 10.2174/1389201043376689. View

19.

Kim D, Joyce G . Cross-catalytic replication of an RNA ligase ribozyme. Chem Biol. 2004; 11(11):1505-12. DOI: 10.1016/j.chembiol.2004.08.021. View

20.

Mitra S, Hanson D, Schlaepfer D . Focal adhesion kinase: in command and control of cell motility. Nat Rev Mol Cell Biol. 2005; 6(1):56-68. DOI: 10.1038/nrm1549. View