organization, cell adhesion and cell proliferation. Interestingly, the cellular effects of P61-E7 observed pointed to the possible involvement of RhoA in GGTI-mediated cellular effects. First, the above results on the effects of GGTI to induce p21CIP1/WAF1 are consistent with previous studies that have shown that small GTPase protein RhoA (RhoA-GG) acts as a negative regulator of p21CIP1/WAF1 expression [38,39]. Effects of GGTI on actin cytoskeleton also support the possibility that P61-E7 induces cellular effects by blocking geranylgeranylation of RhoA, thereby disrupting its subcellular localization and inhibiting its functions. Indeed, results from our RhoA-GTP pull-down assay indicated that P61-E7 inhibited activation of RhoA in a dose-dependent manner (Figure S3). To assess the implication of RhoA in P61-E7 induced phenotypes, we constructed Panc-1 stable cell line expressing farnesylated RhoA (RhoA-F). Using site-directed mutagenesis, a RhoA-F mutant was generated by altering the CAAX from CLVL to CLVS sequence to render RhoA a substrate of farnesyltransferase. Previous studies showed that farnesylated RhoA has the same subcellular localization as geranylgeranylated RhoA, and that both RhoA prenyl isoforms have similar effects on cell morphology, actin organization, vinculin distribution, cell proliferation and p21CIP1/WAF1 expres- sion [40]. However, unlike wild-type RhoA (RhoA-GG), RhoA-F is expected to be resistant to GGTI treatment. Therefore, if P61- E7 does indeed increase p21CIP1/WAF1 expression by blocking geranylgeranylation of RhoA, stable expression of RhoA-F is expected to overcome P61-E7’s effect on p21CIP1/WAF1 expression. We established populations of Panc-1 cells stably expressing either 3xHA-tagged wild-type RhoA (3xHA-RhoA-GG) or 3xHA-RhoA mutant (3xHA-RhoA-F) as described in ‘‘Materials and Methods,’’ and confirmed the prenylation of 3xHA-RhoA- GG and 3xHA-RhoA-F in these stable cell lines by treating them with DMSO, FTI BMS-225975, or GGTI-298 which is a commercially available GGTI. Treatment with GGTI-298 inhibited protein geranylgeranylation as indicated by the appearance of the unprenylated Rap1 band (Figure 6A, left and right panels). Moreover, as expected, GGTI-298 treatment slowed the mobility of 3xHA-RhoA-GG on SDS-PAGE, indicating geranylgeranylation of 3xHA-RhoA-GG was inhibited (Figure 6A, right panel). However, GGTI-298 did not change the mobility of 3xHA-RhoA-F protein (Figure 6A, left panel). Consistent with observations discussed above (Figure 2C), FTI BMS-225975 inhibited farnesylation of HDJ2, a known substrate of FTase, as shown by the appearance of the slower migrating form of HDJ2 on SDS-PAGE (Figure 6A, left and right panels). Furthermore, treatment with the FTI inhibited farnesylation of 3xHA-RhoA-F (Figure 6A, left panel) while it had no effect on the mobility of 3xHA-RhoA-GG (Figure 6A, right panel). We also checked the expression levels of RhoA-GG and RhoA-F in the Panc-1 stable cell lines used in these experiments. As indicated in Figure 6B, the levels of RhoA-GG and Rho-F were similar in these stable cell lines. To investigate whether stable expression of RhoA-F prevents P61-E7 induction of p21CIP1/WAF1, Panc-1 cells and Panc-1 cells stably expressing either the wild-type RhoA (3xHA-RhoA-GG) or mutant RhoA (3xHA-RhoA-F) were treated with DMSO or various concentrations of P61-E7. Consistent with previous observations, increases in p21CIP1WAF1 protein levels were observed in Panc-1 cell and Panc-1 cells stably expressing 3xHA-RhoA-GG (Figure 6C). On the contrary, induction of p21CIP1/WAF1 was not observed in Panc-1 cells stably expressing 3xHA-RhoA-F (Figure 6C). These results indicated that stable expression of 3xHA-RhoA-F in Panc-1 prevented induction of p21CIP1/WAF1 by P61-E7. Novel Inhibitor of Geranylgeranyltransferase-I We also found that the expression of RhoA-F prevented induction of p27Kip1 protein level by P61-E7. We treated Panc-1 cells and Panc-1 cells stably expressing either 3xHA-RhoA-GG or 3xHA-RhoA-F with DMSO or P61-E7. Consistent with previous observations, increases in p27Kip1 protein levels were observed in Panc-1 cell and Panc-1 cells stably expressing 3xHA-RhoA-GG (Figure 6D). On the other hand, P61-E7 did not increase p27Kip1 protein level in Panc-1 cells stably expressing 3xHA-RhoA-F (Figure 6D). These results showed that stable expression of 3xHA- RhoA-F in Panc-1 inhibited the ability of P61-E7 to increase p27Kip1 level. Next, we investigated whether stable expression of farnesylated RhoA confers resistance to the antiproliferative effect of P61-E7. Following treatment with DMSO or 5 mM of P61-E7, cell proliferation of Panc-1 cells, Panc-1 cells stably expressing 3xHA-RhoA-GG, and 3 different clones of Panc-1 cells stably expressing 3xHA-RhoA-F with different expression levels (Figure 7B) was measured as described previously [20]. As shown in Figure 7A, stable expression of 3xHA-RhoA-F promotes partial resistance to the anti-proliferative effect of P61-E7 in a manner that depends on its expression level in Panc-1 cells. With the highest expression level of RhoA-F, Panc-1 (3xHA-RhoA-F) Clone No. 8 showed the highest resistance to the anti-proliferative effect of P61-E7 when compared with Panc-1 (3xHA-RhoA-F) Clone No. 1 (lowest expression level) and Clone No. 4 (intermediate expression level). These findings suggested that P61-E7 inhibition of cell proliferation in Panc-1 cells is due, in part, to inhibition of RhoA geranylgeranylation and hence its function. We also examined whether cell morphology and actin cytoskeleton changes induced by P61-E7 can be rescued by the stable expression of 3xHA-RhoA-F in Panc-1 cells. As shown in Figure 8, Panc-1 cells expressing 3xHA-RhoA-GG responded to P61-E7, and morphological changes and actin cytoskeleton disorganization were observed. In addition, vinculin staining showed that focal adhesion formation was inhibited by the GGTI treatment. In contrast, P61-E7 failed to induce morphological changes and actin cytoskeleton changes in Panc-1 cells expressing HA-RhoA-F. Furthermore, focal adhesions were still detected even after P61-E7 treatment in this cell line. These results suggest that stable expression of RhoA-F rescues Panc-1 cells from cell rounding and inhibition of focal adhesion formation caused by P61-E7. Discussion In this paper, we report successful identification of a novel GGTI compound P61-E7 that has a tetrahydropyridine scaffold. P61-E7 was derived by converting the free carboxylic acid moiety on the tetrahydropyridine ring of a parent compound P3-E5 to L- leucine amido methyl ester. P61-E7 exhibits increased potency to inhibit protein geranylgeranylation compared to our previous preclinical compound P61-A6 [12,20]. Inhibition of geranylger- anylation was first shown by detecting the appearance of unprenylated Rap1. In addition, P61-E7 was shown to be more effective at inducing cytoplasmic accumulation of RalA and RhoA in comparison to P61-A6. The availability of a compound with increased potency enabled us to define cellular consequences of inhibiting protein geranylger- anylation. By using two compounds with different levels of potency to inhibit geranylgeranylation (P61-E7 and P61-A6), we could look for cellular effects that correlate with the inhibition of geranylger- anylation. We examined the effects of P61-E7 on pancreatic cancer cells Panc-1. We have shown that P61-E7 causes more pronounced cell cycle block at the G1 phase compared with P61- PLoS ONE | www.plosone.org 8 October 2011 | Volume 6 | Issue 10 | e26135