In vivo apoptosis detection with radioiodinated Annexin V in LoVo tumour-bearing mice following Tipifarnib (Zarnestra, R115777) farnesyltransferase inhibitor therapy Bart Cornelissen a, T , Christophe Lahorte a , Veerle Kersemans a , Gabriela Capriotti b , Elena Bonanno c , Alberto Signore b , Christophe Van de Wiele d , Rudi A. Dierckx d , Guido Slegers a a Laboratory of Radiopharmacy, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium b Nuclear Medicine Unit, Policlinico Umberto I, Viale del Policlinico 155, Rome, Italy c Anatomic Pathology, Tor Vergata Institute, University of Rome, Viale Oxford, 81 000133 Rome, Italy d Division of Nuclear Medicine, Ghent University Hospital, De Pintelaan 185, Ghent, Belgium Received 24 August 2004; revised 9 December 2004; accepted 3 January 2005 Abstract In this paper, the use of 123 I-Annexin V for the detection of farnesyltransferase inhibitor (FTI)-induced apoptosis in tumour-bearing athymic mice is described. In vitro binding assays on LoVo cells show time- and dosage-dependent 125 I-Annexin V binding upon treatment with Tipifarnib (Zarnestra, R115777), a selective and potent FTI. In vivo experiments using planar gamma scintigraphy on LoVo inoculated mice show a 40% increased 123 I-Annexin V uptake 8 h after a single oral administration of 100 mg/kg Tipifarnib in 20% h-cyclodextrin in 0.1 M HCl, as well as after 3 days of twice daily treatments with the same dose. Ex vivo TUNEL assays, detecting end-stage apoptotic cells, correlate significantly with both in vitro and in vivo results. The percentage of necrosis is also increased by Tipifarnib treatment, but is too low to interfere with the 123 I-Annexin V uptake. It can be concluded that 123 I-Annexin V can be used to monitor Tipifarnib-induced apoptosis in LoVo xenograft tumours in athymic mice. Future applications might include the early prediction of FTI response and the selection of FTI-sensitive patients very shortly after treatment initiation. Subsequently, such patients would greatly benefit from a noninvasive and fast therapy evaluation. D 2005 Elsevier Inc. All rights reserved. Keywords: Annexin V; Apoptosis; Farnesyl transferase inhibitor; Oncology; Tipifarnib 1. Introduction Farnesyltransferase inhibitors (FTIs) are a newly developed class of anti-tumour agents and were originally designed to inhibit the posttranslational farnesylation of the Ras protein, a key regulator of many growth regulating signalling pathways [1,2]. Recent studies, however, demonstrated that Ras was not the only target of the FTIs. Other possible molecular targets, such as RhoB, the phosphoinositol pathway and the centromeric proteins CENP-E and CENP-F, have been proposed [3–8]. Nonetheless, the exact mechanism of action of the FTIs remains unclear. FTIs exhibit many different effects on mammalian cells of which (soft agar) growth inhibition is the most important. Other influences include the following: (i) mitotic cell cycle arrest in the G0/G1 phase or in the G2/M phase, dependent on the type of cell line; (ii) influence on the actin and tubulin cytoskeleton; and (iii) the induction of apoptosis or programmed cell death [9,10]. The induction of apoptosis by FTIs has been reported by many researchers. FTI-induced programmed cell death in vitro by the selective FTIs SCH56582 and L-744,832 was observed in v-K-Ras transformed normal rat kidney (NRK) cells and H-Ras transformed rat embryonic fibroblast Rat-1 cells, respectively [11,12]. Beaupre et al. [13] showed that Tipifarnib (Zarnestra, R115777) is able to induce apoptosis in myeloma cells via Ras-independent pathways. End et al. [14] demonstrated the importance of in vivo apoptosis effects in growth inhibition experiments of Tipifarnib on 0969-8051/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nucmedbio.2005.01.001 T Corresponding author. Tel.: +32 9 264 80 45; fax: +32 9 264 80 71. E-mail address: bart.cornelissen@ugent.be (B. Cornelissen). Nuclear Medicine and Biology 32 (2005) 233 – 239 www.elsevier.com/locate/nucmedbio