Innovative (Survivin-based) or (p21-based) cell culture screening system for highly cell-permeable PAK1-blockers

Several years ago, a unique adenoviral construct called “Ad-Surv-GFP” was developed originally in order to detect bladder cancers in urine of patients (1). This construct carries a fusion gene in which the promoter region (around 250 bp) of survivin gene is fused to cDNA of GFP, so that GFP is expressed under the control of survivin gene promoter. When bladder carcinoma cells are infected with this virus, GFP is expressed at a very high level, while the infected normal cells express GFP only at a very low level (1).  According to both our recent unpublished observation (2, the surviving-suppressor YM155 blocks PAK1 in cell culture) and the down-regulation of survivin expression in PAK1-deficient mice (3), it is now clear that PAK1 is essential for the activation of survivin gene promoter (Surv). Thus, in principle, this adenoviral construct or more preferably a mammalian expression vector harbouring Surv-LacZ fusion gene could be used for the GFP/LacZ-based screening of potent (highly cell-permeable) PAK1-blockers in cell culture of RAS-transformants such as A549 lung cancer cells and pancreatic cancer cells where PAK1 is abnormally activated. In general, "LacZ" signal is more readily detectable than "GFP" signal.

Interestingly, COX-2 gene promoter also requires PAK1 for its activation (4). Thus, there remains  another  possibility that COX-2-LacZ vector also could be used to screen for PAK1-blockers in cell culture. The most critical “technical” question is which promoter is more sensitive to PAK1 or PAK1-blockers. 

Alternatively, p21/WAF (CDK inhibitor) gene promoter could be used as a positive indicator for anti-PAK1 activity, because more than a decade ago we have shown that PAK1 down-regulates selectively the p21 expression (5). Unfortunately, however, "p21 promoter-LacZ " fusion construct has not been commercially available. However, recently I found that a few groups have used either "p21-promoter-LacZ (or -Luciferase) " constructs for cells or whole mice. That is very encouraging! 

In C. elegans (CL2070), we have successfully used “HSP16.2 gene promoter-GFP” fusion construct as a “positive” indicator for anti-PAK1 activity, and PAK1-blockers such as ARC and CAPE from propolis indeed boost the GFP expression in this worm (6).

References: 

 

1. Murali A, Kasman L, Voelkel-Johnson C. Adenoviral infectivity of exfoliated viable cells in urine: implications for the detection of bladder cancer. BMC Cancer. 2011; 11: 168.
2.  Nguyen BC, Ahn MR, Tawata S. Maruta H. YM155, the survivin suppressor, blocks the oncogenic/ageing kinase PAK1, and inhibits the migration of HUVECs in cell culture. manuscript in preparation.
3. Chen YC, Fueger PT, Wang Z. Depletion of PAK1 enhances ubiquitin-mediated survivin degradation in pancreatic beta-cells. Islets. 2013 ; 5: 22-8.
4. R Wu, AL. Abramson, MH. Symons, BM. Steinberg. Pak1 and Pak2 are activated in recurrent respiratory papillomas, contributing to one pathway of Rac1-mediated COX-2 expression. Int J Cancer. 2010, 127, 2230-7. 
5. Nheu T, He H, Hirokawa Y, Walker F, Wood J, Maruta H. PAK is essential for RAS-induced upregulation of cyclin D1 during the G1 to S transition. Cell Cycle. 2004 ; 3: 71-4
6. Yanase, S； Luo Y；Maruta H. PAK1-deficiency/down-regulation reduces brood size, activates HSP16.2 gene and extends lifespan in C. elegans. Drug Discov. Ther. 2013, 7 (1), 29–35.