Using Apoptosis for Targeted Cancer Therapy by a New Gonadotropin Releasing Hormone-DNA Fragmentation Factor 40 Chimeric Protein Ahmi Ben-Yehudah, Rami Aqeilan, Dana Robashkevich, and Haya Lorberboum-Galski 1 Department of Cellular Biochemistry and Human Genetics, Hebrew University–Hadassah Medical School, Jerusalem 91120, Israel ABSTRACT Purpose: GnRH-based chimeric proteins have been shown to specifically target and kill adenocarcinomas both in vitro and in vivo. The purpose of this study is to construct a new GnRH-based chimeric protein for the treatment of adenocarcinomas in humans. Results: In this study, we constructed and characterized a new chimeric protein, GnRH-DFF40, composed of a new human killing moiety: the apoptotic DNase-DFF40 (DNA fragmentation factor), known also as caspase-activated DNase (CAD). GnRH-DFF40 exhibits DNase activity in vitro. We found that this chimeric protein can target and kill adenocarcinoma cells. Such death occurs via apoptotic path- ways, resulting in an increase in the sub-G 1 population, DNA fragmentation, terminal deoxynucleotidyl transferase- mediated nick end labeling (TUNEL)-positive cells, and morphology typical of apoptotic cells. These apoptotic events involve the mitochondria because we found cytochrome c depletion and caspase-9 and caspase-3 activation. Prelimi- nary in vivo results showed that treatment of colon adeno- carcinoma xenografts in nude mice with the new chimeric protein caused a reduction in tumor weight. Conclusions: Because GnRH-DFF40 is a whole human- based chimeric protein when applied to humans, the non- specific toxicity and immunogenicity seen with bacterial/ plant-based chimeric proteins should be avoided. Thus, GnRH-DFF40 is a promising candidate for the treatment of adenocarcinomas in humans. INTRODUCTION We recently described chimeric proteins that can both target and kill adenocarcinoma cells (1–3). These chimeric proteins include GnRH, 2 also known as luteinizing hormone releasing hormone, as the targeting domain. GnRH is a hypo- thalamic decapeptide that plays a role in the reproductive system (4). For the killing domain, we used various derivatives of PE (1). This bacterial toxin inhibits protein synthesis in eukaryotic cells, thereby causing cell death (5, 6). GnRH-PE chimeric proteins kill a wide variety of cancers arising from hormone- responsive tissues, as well as from nonresponsive ones such as ovarian, breast, endometrial, cervical, colon, lung, hepatic, and renal carcinomas, all confined to the adenocarcinoma type (1– 3). PE and other bacterial or plant toxins are commonly used in chimeric proteins (7). Although several chimeric proteins are already in clinical use, they have several disadvantages that limit their clinical application. The major obstacle in the clinical application of such chimeric proteins is the human immune response they elicit, mainly toward the toxin moiety. Bacterial toxins such as PE are highly immunogenic and cannot be humanized by standard techniques. Moreover, each chimeric protein displays some degree of nonspecific toxicity (8, 9). To overcome these obstacles, we propose the use of human apoptosis-inducing proteins as the killing moiety. Apoptosis, or programmed cell death, is an intrinsic mech- anism common to all cells. Initially found in Caenorhabditis elegans (10, 11), there are three protein families involved in promoting or inhibiting apoptosis: the Bcl-2 family (12, 13); Apaf-1 (14, 15); and the caspases (16 –18). The first chimeric protein constructed with an apoptotic moiety as the trigger of selective cell death was IL-2-Bax (19). In this chimera, the killing domain is Bax, a proapoptotic protein of the Bcl-2 family. IL-2-Bax specifically targets and kills cells overexpressing the IL-2 receptor via apoptosis (19). Next, we constructed a number of GnRH-based chimeric proteins fused to proapoptotic proteins Bax, Bik, and Bak of the Bcl-2 family (20). These proteins are able to specifically target and kill adenocarcinoma cells, similar to the specific activity of GnRH-PE chimeric proteins (1–3). However, as we didn’t know which apoptotic protein will serve as the most potent killing domain in the chimeric protein, we have chosen an executor of apoptosis, a nuclease that is a downstream component of the complexed apoptotic cascade and tested its potential as a novel killing domain of a GnRH-based chimeric protein. Our present study focuses on the DFF40 apoptotic protein as the novel killing domain of a chimeric protein and examines Received 7/12/02; revised 10/30/02; accepted 11/1/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Department of Cellular Biochemistry and Human Genetics, Hebrew University–Hadassah Medical School, Jerusalem 91120, Israel. Phone: 972-2-6757465, Fax: 972-2-6415848; E-mail: hayalg@md2.huji.ac.il. 2 The abbreviations used are: GnRH, gonadotropin releasing hormone; PE, Pseudomonas exotoxin A; IL, interleukin; DFF, DNA fragmentation factor; FACS, fluorescence-activated cell sorting; TUNEL, terminal deoxynucleo- tidyl transferase-mediated nick end labeling; DAPI, 4',6-diamidino-2- phenylindole; NLS, nuclear localization sequence. 1179 Vol. 9, 1179 –1190, March 2003 Clinical Cancer Research Research. on April 12, 2016. © 2003 American Association for Cancer clincancerres.aacrjournals.org Downloaded from