Biochemical and Biophysical Research Communications
A novel model to identify interaction partners of the PTEN tumor suppressor gene in human bladder cancer
Section snippets
Recombinant DNA techniques
PTEN inducible system. Humanized LacI and the inducible SV40 promoter have been described elsewhere [3]. The LacI cDNA was cloned into pIRESpuro2 (Clonetech, Mountain View, CA, USA) at the EcoRI site. The CMV promoter in pcDNA3.1zeo (Invitrogen, Carlsbad, CA, USA) was cut out with NheI and NruI and substituted with the LacI suppressed SV40 promoter. PTEN with a C-terminal hemagglutinin (HA) tag [4] was cloned at the EcoRI site in this vector.
Cloning putative PTEN-binding partners. All tested
IPTG induces PTEN protein expression at physiological levels and alters AKT phosphorylation
To facilitate biologically relevant PTEN-binding partner discovery, an inducible expression system was constructed (Fig. 1A) in the PTEN null human bladder cancer cell line UMUC-3. PTEN-HA fusion protein was expressed under the control of an IPTG inducible SV40 promoter and single cell clones were created through limiting dilutions. These were screened for PTEN-HA induction by Western blot analysis (Fig. 1B). Maximum induction was reached at 1 mM IPTG and further increased concentration did not
Discussion
The study of tumor suppressors is complicated by the fact that their exogenous introduction in a cancer cell population in vitro is susceptible for the direct selection against transfected cells by the very nature of the gene product. One way of circumventing this problem is to utilize an inducible system regulating the expression of the transgene. Here we modified the inducible SV40/humanized LacI system previously developed for transgenic mouse models [3]. The system has advantages for this
Acknowledgments
The authors acknowledge the expert technical assistance of Jennifer Bryant and Charles Owens. The humanized LacI cDNA and the inducible SV40 promoter, as well as valuable advice on in vivo induction, were supplied by Dr. Heidi Scrable. This work was supported by NIH Grant GM37537 to D.F.H. and CA075115 to D.T.
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