1. The early testosterone-induced actin reorganization in colon cancer is independent of iAR signalling control 15min30min60min + TAC control 15min 30min60min + TAC GFGFGFGF n=5 G/ F ratio % * ** 20 40 60 80 control15 min30 min60 min Fig.1. (A) 24h serum-starved cells were stimulated with 10 -7 M testosterone-HSA. Cells were stained with rhodamine-phalloidin for filamentous actin and DRAQ5™ for nuclei and subsequently analysed by confocal laser scanning microscopy. (B) G- and F- actin were measured by quantitative immunoblot analysis after Triton X-100 subcellular fractionation. Bars present the G/F actin mean value  SE of n=5 independent duplicate experiments (** P < 0.01, * P<0.05). 15min 30min 60min iAR- siRNA + TAC control Flutamide + TAC 15min30min60mincontrol GF GF GFGF 15min 30min 60min flutamide+TAC actin iAR neg siRNA iAR siRNA control fold decrease 1.00.87±0.110.28±0.02 ** n=3 Fig.2. 24h serum-starved cells were stimulated with 10 -7 M testosterone- HSA in the presence or absence of 10 -6 M flutamide. Cells were stained with rhodamine-phalloidin for filamentous actin and DRAQ5™ for nuclei and subsequently analysed by confocal laser scanning microscopy. Fig.3. 24h serum-starved cells were stimulated with 10 -7 M testosterone- HSA were silenced with or without siRNA against iAR for the indicated time. Cells were stained with rhodamine-phalloidin for filamentous actin and DRAQ5™ for nuclei and subsequently analysed by confocal laser scanning microscopy. Summary: Recently it has been reported that testosterone conjugates trigger rapid actin cytoskeleton reorganization in colon tumour cells. This effect was recognized as a critical initial event, controlling apoptosis and inhibiting cell migration. The present study addressed whether these early effects in colon tumours are controlled by the classical androgen receptor (iAR) and/or through the activation of the newly identified membrane androgen receptors (mAR). In line with previous observations, treatment of Caco2 colon cancer cells with testosterone albumin conjugates (TAC) induced rapid (within 15 minutes) actin cytoskeleton restructuring. This was evident from confocal laser microscopic analysis showing clear formation of newly synthesized actin filaments, while quantitative determinations of the G to F- actin ratio fully confirmed the rapid actin polymerization. The same experiments were then performed in the presence of the specific nonsteroidal antiandrogen flutamide or in cells transfected with a specific siRNA targeting iAR that resulted in reduced expression of iAR. Upon blocking of iAR either by flutamide or by siRNA silencing actin polymerization was still induced in Caco2 cells. These experiments underlined that the rapid testosterone-induced actin polymerization is not correlated with the classical androgen receptor signalling and imply the involvement of early mAR signalling in testosterone-treated colon cancer cells. Introduction: Early actin cytoskeleton reorganization is an important initial step of the non-genomic steroid hormone actions of androgens in a variety of tumor cell models [1-3]. Actin restructuring modifies cell- substratum adhesion and has been reported to control many aspects of cellular physiology, including cell motility, secretion, apoptosis, cell proliferation and survival.From these and many other reports it became widely accepted that modifications in microfilament dynamics triggered by membrane receptor signaling play a pivotal role in various cell responses and seem to be involved in a large number of human diseases [4]. However, the potential involvement of the classical androgen receptor (iAR) in the early actin reorganization has not been addressed. In the present study we have analyzed the rapid actin restructuring in the presence of the specific nonsteroidal antiandrogen flutamide and cells transfected with a specific siRNA targeting iAR that resulted in reduced expression of iAR. Our results indicate specific and rapid testosterone effects on actin reorganiztion that are independent of iAR signaling. Conclusions: Activation of membrane Androgen Receptors (mAR) induces rapid actin cytoskeleton reorganization. Control experiments using either the anti-androgen flutamide or appropriate siRNAs to silence iAR did not influenced the rapid actin polymerization. These findings imply that actin reorganization is stimulated directly by the membrane androgen binding sites. Accordingly, the rapid actin polymerization triggered by testosterone albumin conjugates (TAC) is not correlated with the classical androgen receptor signaling. Saad Alkahtani Department of Biology, Teachers College, King Saud University, Riyadh, Saudi Arabia. References: [1] S. Gu, N. Papadopoulou, E.M. Gehring, O. Nasir, K. Dimas, S.K. Bhavsar, M. Foller, K. Alevizopoulos, F. Lang, C. Stournaras, (2009) Functional membrane androgen receptors in colon tumors trigger pro-apoptotic responses in vitro and reduce drastically tumor incidence in vivo, Mol Cancer. 8 114. [2] N. Papadopoulou, I. Charalampopoulos, K. Alevizopoulos, A. Gravanis, C. Stournaras, (2008) Rho/ROCK/actin signaling regulates membrane androgen receptor induced apoptosis in prostate cancer cells, Exp Cell Res. 314 3162-3174. [3] E.A. Papakonstanti, M. Kampa, E. Castanas, C. Stournaras, (2003) A rapid, nongenomic, signaling pathway regulates the actin reorganization induced by activation of membrane testosterone receptors, Mol Endocrinol. 17 870-881. [4] F.M. Vega, A.J. Ridley, (2008) Rho GTAases in cancer cell biology, FEBS Lett. 582 2093-2101. Results: In line with previous observations [1], treatment of Caco2 colon tumor cells with testosterone albumin conjugates (TAC) induced rapid (within 15 minutes) actin cytoskeleton restructuring. This was evident from confocal laser microscopic analysis showing clear formation of newly synthesized actin filaments (Fig. 1 A), while quantitative determinations of the G to F-actin ratio fully confirmed the rapid actin polymerization (Fig. 1B). To identify the potential involvement of the classical androgen receptor (iAR) in this early event, the same experiments were performed in the presence of the specific nonsteroidal antiandrogen flutamide (Fig 2), or in cells transfected with a specific siRNA targeting iAR that resulted in reduced expression of iAR (Fig 3). As shown in Figures 2 and 3, upon blocking of iAR either by flutamide or by siRNA silencing actin polymerization was still induced in Caco2 cells treated by testosterone conjugates, indicating that iAR is not involved in the early actin reorganization. These findings are in line with previous reports, which established clearly that neither flutamide treatment nor iAR silencing inhibited any of the mAR responses tested in prostate cancer cells [3]. Fig.4. Confocal laser scanning microscopy of Caco-2 cells treated with either 10 -6 M flutamide (left panels) or iAR siRNA (right panels) and stained with testosterone-HSA-FITC. Magnification ×100.. TAC-FITCHSA-FITCTAC-FITCHSA-FITC Flutamide treated Caco-2 cellsiAR silenced Caco-2 cells TAC-FITC control A B