Nuclear drug delivery to target tumour cells

European Journal of Pharmacology 625 (2009) 174–180

Contents lists available at ScienceDirect

European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Review

Nuclear drug delivery to target tumour cells Kylie M. Wagstaff, David A. Jans ⁎ Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic 3800, Australia

a r t i c l e

i n f o

Article history: Received 10 May 2009 Received in revised form 5 June 2009 Accepted 22 June 2009 Available online 15 October 2009 Keywords: Cancer Drug delivery Nuclear import Tumour cell-specific Gene therapy Nuclear localisation signal

a b s t r a c t Cancer remains one of the leading causes of death worldwide. Although enticing, the concept of a chemotherapeutic treatment directed towards a single target that kills tumour cells, without any harmful side effects or death of neighbouring cells is probably naïve, due to the fact that tumour cells arise from normal cells and share many common biological features with them. Various means to damage/destroy tumour cells preferentially have been developed, but as yet, none are truly selective. However, by combining numerous tumour-specific/-enhanced targeting signals into a single modular multifaceted approach, it may prove possible some time in the future to achieve the desired outcome, without any unwanted bystander effects, with the delivery of cytotoxic drugs/DNA directly to the nucleus specifically within tumour cells of great interest in this context. To achieve this, modules such as the tumour-specific nuclear targeting signal of the chicken anemia virus Apoptin protein represent exciting possibilities. The present review discusses the question of nuclear delivery of bioactive molecules and its associated problems, as well as recent progress towards the development of tumour-specific modular recombinant transporters as viable anti-cancer therapeutics. In particular we focus upon the incorporation of tumour cell-selective nuclear targeting into these systems as a means to deliver cytotoxic genes or drugs to the very heart of tumour cells. © 2009 Elsevier B.V. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nuclear delivery of drugs: Lessons learned from viruses . . . . . . . . . . . . . . . . . . . . . . 2.1. Cellular barriers to nuclear delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Nuclear transport mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Regulation of nuclear transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Generating effective viral mimics: A modular approach . . . . . . . . . . . . . . . . . . . 3. NLSs are the key to efficient nuclear delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. The importance of drug delivery to the nucleus . . . . . . . . . . . . . . . . . . . . . . . 3.2. Tumour cell-specific nuclear targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. The C-terminus of Apoptin represents a tumour cell-enhanced nuclear targeting signal 4. A new class of modular transporters with multifactorial targeting systems: The tNTS at the forefront? . . 5. Towards the future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Despite billions of research dollars spent globally each year, cancer remains one of the leading causes of death worldwide, both in first world nations like the United States of America, where it remains the second leading of cause of death, but also in developing countries (Weir et al., 2003). Although each form of cancer is subtly different, ⁎ Corresponding author. E-mail address: [email protected] (D.A. Jans). 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.06.069

. . . . . . . . . . . . .

. . . . . . . . . . . . .

. . . . . . . . . . . . .

. . . . . . . . . . . . .

. . . . . . . . . . . . .

. . . . . . . . . . . . .

. . . . . . . . . . . . .

. . . . . . . . . . . . .

. . . . . . . . . . . . .

. . . . . . . . . . . . .

. . . . . . . . . . . . .

. . . . . . . . . . . . .

. . . . . . . . . . . . .

. . . . . . . . . . . . .

. . . . . . . . . . . . .

. . . . . . . . . . . . .

. . . . . . . . . . . . .

. . . . . . . . . . . . .

. . . . . . . . . . . . .

174 175 175 175 176 176 176 177 177 177 177 179 179

cancer can generally be regarded as being the result of accumulated and/or inherited genetic abnormalities, leading to prolonged cell survival, resistance to normal cellular death and an increased propensity to proliferate. Despite the fact that our understanding of the molecular mechanisms underpinning many cancers has progressed rapidly, our ability to treat the disease itself remains extremely limited. Most currently utilised cancer therapeutics rely upon the successful eradication of tumour cells from the patient by the therapeutic agent; unfortunately this almost always results in the unnecessary and unwanted death of surrounding “normal” bystander

K.M. Wagstaff, D.A. Jans / European Journal of Pharmacology 625 (2009) 174–180

cells (Guillemard and Saragovi, 2004). Moreover, due to the nature and mechanisms of action of many if not most of these drugs, it is the highly proliferating normal cells of the body that tend to be affected the most, including cells within the bone marrow and the immune system in general, with the end result being a severely immunocompromised patient who is then highly susceptible to invasion by potentially life-threatening pathogens. Thus, it is clear that a new generation of highly efficient and above all specific anti-cancer agents are urgently required. One potential avenue for the generation of these “super” drugs is through specific targeting of highly vulnerable sites within tumour cells themselves, with one of the most attractive being the nucleus. Here we discuss the possibility for using recently identified tumour cell-enhanced nuclear targeting signals (tNTS) to deliver high levels of suicide genes or drugs to the very heart of a tumour cell, without affecting healthy neighbouring cells. This, together with a combinatorial or modular approach to anti-cancer therapeutic delivery mechanisms appears likely to be effective in treating cancer specifically as well as efficiently (Alvisi et al., 2006). 2. Nuclear delivery of drugs: Lessons learned from viruses One of the most effective proposed methods to effect efficient tumour cell killing is to target drugs specifically to tumour cells as opposed to normal cells, and then subsequently to direct them to hypersensitive subcellular sites within these cells, such as the nucleus (Alvisi et al., 2006). This should help to accomplish the ultimate goal of being able to avoid harmful side effects on the surrounding normal healthy cells, without compromising on cytotoxicity towards tumour cells. However, in order to achieve this, one needs to overcome a series of natural cellular barriers (see Fig. 1), which prevent the entry of foreign material into all cells and more importantly into the nucleus within these cells (Khalil et al., 2006; Wagstaff and Jans, 2007). Viruses have evolved specific mechanisms to overcome such barriers, with many relying on the delivery of the viral genome into the nucleus of host cells as an essential part of their life cycle (Robbins and Ghivizzani, 1998). However, safety considerations such as pathogenicity, oncogenicity and the stimulation of an immune response have hampered their use in clinical settings. To overcome these issues,

Fig. 1. Cellular barriers to bioactive molecules/nanocarriers. Schematic of the cellular barriers faced by bioactive molecules such as drugs or therapeutic DNA en route to the nucleus. The plasma membrane represents the first major barrier to bioactive compounds and can be overcome through the use of ligands to target cell-surface receptors, resulting in an endocytotic method of uptake (1) or through direct entry mechanisms such as the use of protein transduction domains or electroporation (2). Once inside the cell escape from endosomes is necessary (3), if taken up by endocytosis, in order to escape degradation (4), whilst the numerous degradative enzymes in the cytosol must also be avoided (5). To access the nucleus, transport through the nuclear pore complexes (NPC) embedded in the nuclear envelope (6) is necessary, to enable ultimate accumulation of the drug/DNA within the nucleus (7), to lead to a biological effect (8).

175

much progress has been made in the development of viral mimics or modular recombinant transporters (MRTs) that imitate viruses by replicating/retaining all of the necessary cellular and subcellular targeting functions of the virus as a whole, but without the associated safety concerns (Glover et al., 2005). 2.1. Cellular barriers to nuclear delivery The first major barrier to nuclear drug delivery is the plasma membrane, which surrounds all mammalian cells and restricts the passage of large hydrophilic or charged molecules (Khalil et al., 2006; Wagstaff and Jans, 2006b, 2007). Thus, the first step in drug delivery is cell entry, which must involve binding to and passage through the cellular membrane. To facilitate this, many viruses recognise specific receptors expressed/exposed on the extracellular surface of target cells. Depending on the virus or vector in question, receptor binding may result in subsequent internalisation into an endosome, as is the case for non-enveloped DNA viruses such as adenovirus (Seth, 1994). Endosomal entrapment ultimately results in degradation of the enclosed ligand when the endosome fuses with a lysosome (Conner and Schmid, 2003). To avoid this, many viral proteins also function as endosomal escape moieties, undergoing conformational changes in response to the decreased pH found in endolysosomes, thus disrupting the vesicle membrane and facilitating release of the virus into the cytoplasm (Alvisi et al., 2006; Seth, 1994). The virus must subsequently traverse the cytoplasm through the crowded network of the cytoskeleton, avoid degradation, and translocate into the nucleus in order to be expressed and replicated (Wagstaff and Jans, 2007), or in some cases, integrated into the host cell genome, as is the case for retroviruses. Nuclear transport is known to be the most rate-limiting step in this process, implying that the nuclear envelope represents the most substantial barrier for gene or drug delivery to cells, including cancer cells (Vaughan et al., 2006). 2.2. Nuclear transport mechanisms The nucleus is the control centre of the eukaryotic cell, being both the site of storage and replication of the cell's genetic material, and of processes such as transcription and ribosome assembly that are central to synthesising the cellular complement of proteins that carry out all of its functions. The nucleus is separated from the rest of the cell cytoplasm by the double membrane structure of the nuclear envelope, which is punctated by nuclear pore complexes, through which all transport between the nucleus and cytoplasm occurs (Harel and Forbes, 2004; Wente, 2000). The nuclear pore complex has a tripartite structure, consisting of a central channel and a cytoplasmic and nuclear ring, which are made up of more than 50 different proteins known as nucleoporins (Wente, 2000). Each individual nuclear pore complex is the location of approximately 1000 translocation events every second, in either direction, consisting of both active and passive events (Ribbeck and Gorlich, 2001). Small molecules are able to pass through the nuclear pore complex via passive diffusion, but molecules N∼45 kDa require specific targeting signals to gain either access to or egress from the nucleus (Gorlich, 1998; Gorlich and Kutay, 1999; Poon and Jans, 2005). In the import direction, nuclear localisation signals (NLSs) are recognised by members of the Importin (also known as karyopherin) superfamily of cellular nuclear transport proteins, of which there are two main types, α and β (Wente, 2000). NLSs themselves fall into one of two categories, classical or non-classical sequences (Jans et al., 1998). The best understood types of classical NLS are those that are typified by the NLS of the simian virus 40 large tumour antigen (T-ag: PKKKRKV132; single letter amino acid code, basic residues in bold), which is a monopartite NLS comprising a single stretch of basic amino acids (Kalderon et al., 1984a,b), or that of the Xenopus protein nucleoplasmin (KRPAATKKAGQAKKKK170), which is bipartite and

176

K.M. Wagstaff, D.A. Jans / European Journal of Pharmacology 625 (2009) 174–180

consists of two short stretches of basic amino acids separated by a 10– 12 amino acid spacer (Robbins et al., 1991). Non-classical NLSs on the other hand, such as the largely hydrophobic 38-residue M9 sequence of the human mRNA-binding protein hnRNP (Pollard et al., 1996) lack runs of basic amino acids. Conventional nuclear transport (Fig. 2) involves recognition of an NLS by the Importin α subunit of the Importin α/β heterodimer (Goldfarb et al., 2004), followed by docking at and translocation through the nuclear pore complex mediated by the Importin β subunit (Bayliss et al., 2000a,b; Wente, 2000). Once inside the nucleus, the monomeric guanine nucleotide binding protein Ran, in its GTP bound form, binds to Importin β to actively displace Importin α and effect release of the cargo into the nucleoplasm (Azuma and Dasso, 2000). Importin β itself and its numerous homologues are also able to mediate nuclear transport of many cargoes in an analogous fashion, but without the need for Importin α (Jakel et al., 1999; Lam et al., 1999; Langer, 2000; Mosammaparast et al., 2002; Mosammaparast and Pemberton, 2004; Pollard et al., 1996; Wagstaff et al., 2007). In humans, N20 homologues of Importin β have been described, whereas 6 Importin αs are known, which differ in both their cargo specificity as well as tissue distribution patterns (Hogarth et al., 2006; Wente, 2000). Nuclear export (Fig. 2) is an analogous process to nuclear import, requiring nuclear export signals (NESs), which are recognised by Imp β homologues known as Exportins, of which Exportin-1 (CRM-1) is the best known example, largely through the fact that its activity can be specifically inhibited by the drug Leptomycin B (Fukuda et al., 1997; Kudo et al., 1998). 2.3. Regulation of nuclear transport Since numerous proteins need to be shuttled into/out of the nucleus in precisely regulated fashion to perform various roles in response to differing stimuli and throughout the cell life cycle, and given that many proteins contain more than one subcellular localisation signal, nuclear transport pathways can be regulated in numerous ways (Poon and Jans, 2005). The best characterised of these regulatory means is through phosphorylation of cargo proteins, usually of sequences close to the NLS/ NES, resulting in either enhanced or diminished affinity for the requisite

Importin/Exportin protein (Jans et al., 2000). The first reported example of regulated nuclear import of a protein was that of T-ag, whose subcellular distribution is regulated by several phosphorylation sites adjacent to the NLS, including a protein kinase 2 recognition site located 14 amino acids upstream (SSDDE115), which when phosphorylated at S111/112 significantly increases the affinity of the T-ag NLS for the Importin α/β heterodimer, resulting in enhanced nuclear import (Hubner et al., 1997). Similar regulatory mechanisms have also been identified in other Importin α/β-recognised NLS-containing proteins (Alvisi et al., 2005; Kaffman et al., 1998; Zhang et al., 2001). Nuclear export of proteins can also be similarly regulated by phosphorylation, such as in the case of FOXO-1, where phosphorylation near to a CRM-1 recognised NES promotes nuclear export by stabilising the FOXO-1/ CRM-1 interaction, or Pho4, where phosphorylation at S114/128 facilitates recognition by Exportin-4, resulting in nuclear export (Komeili and O'Shea, 1999; Zhao et al., 2004). NLS-Importin interactions can also be regulated by other mechanisms, including regulation of Importin expression levels and tissue distribution (for review see (Poon and Jans, 2005)). 2.4. Generating effective viral mimics: A modular approach Ideally, a combination of elements to overcome each of the cells' innate barriers should enable effective drug delivery to be achieved. For tumour cell-specific delivery, these elements should additionally possess selectivity for cancer cells. For example, ligands for cellsurface receptors that are specific to or upregulated on tumour cells can be included as cell targeting moieties in a modular recombinant system. Examples of suitable receptor targets exist, including the Transferrin receptor, which is overexpressed in rapidly dividing cells such as tumour cells, due to the critical need for iron (Wagner et al., 1991,1992), and the Folate receptor, which is upregulated under similar circumstances (Cho et al., 2005). Combination of an endosomal escape moiety, such as the diphtheria toxin translocation domain (Box et al., 2003; Rosenkranz et al., 2003) with an effective NLS, can result in an efficient drug/gene delivery agent. Examples of such modular transporters include those capable of delivering reporter genes or drugs, such as photosensitising agents, specifically to tumour cells in culture (Gilyazova et al., 2006; Sobolev, 2008), to the liver of mice following intravenous injection (Nishikawa et al., 2000), or to the nucleus of non-dividing cells (Glover et al., 2009). 3. NLSs are the key to efficient nuclear delivery

Fig. 2. Nucleocytoplasmic transport. 1. Classical nuclear transport involves the recognition of a nuclear localisation signal (NLS) on a cargo protein by Importin α as part of the Importin α/β heterodimer, followed by docking at and translocation through the nuclear pore complex (NPC) embedded in the nuclear envelope. Once inside the nucleus RanGTP binds to Importin β and dissociates the complex. 2. Importin β alone and many of its homologues can mediate the nuclear transport of various cargoes in a similar fashion, but without the need for Importin α. 3. Nuclear export is an analogous process to nuclear import, whereby a nuclear export sequence (NES) on a nuclear cargo protein is recognised in a trimeric complex by Exportin proteins (Importin β homologues) such as CRM-1 and RanGTP. Docking and translocation through the NPC is mediated by the Exportin and once in the cytoplasm, hydrolysis of RanGTP to RanGDP mediates the dissociation.

It is becoming increasingly clear that nuclear transport of DNA is the key determinant of effective gene delivery (Wagstaff and Jans, 2007), and that increasing nuclear delivery by incorporating an efficient NLS results in significantly higher levels of overall gene expression in most cases (Escriou et al., 2003; Keller et al., 2003; Wagstaff et al., 2007, 2008; Wagstaff and Jans, 2007). We have recently developed an efficient nonviral gene delivery method based upon engineered histones, where optimised nuclear targeting of the constructs through inclusion of an optimised T-ag NLS resulted in significant increased transgene expression (Wagstaff et al., 2007, 2008). Similarly, inclusion of the T-ag NLS within a recombinant modular transporter containing β-galactosidase as a carrier, poly-lysine as a DNA-compacting agent and the foot-andmouth disease virus RGD sequence as a cell targeting moiety (to interact with cell-surface integrin proteins) resulted in a 30-fold increase in transfection efficiency of CaCo2 cells when compared to the transport complex lacking the T-ag NLS (Aris and Villaverde, 2003). Nuclear targeting is especially important for many strategies used in the treatment of cancer cells, including suicide gene therapy (Wagstaff and Jans, 2006b). This form of therapy relies on the use of suicide genes, such as the Herpes simplex virus-1 thymidine kinase gene, which can specifically convert a harmless pro-drug, in this case ganciclovir, into a potent cytotoxic agent (Liu et al., 2001; Tasciotti et al., 2003). Despite the

K.M. Wagstaff, D.A. Jans / European Journal of Pharmacology 625 (2009) 174–180

popularity of this anti-cancer treatment, success has been limited by low expression levels of thymidine kinase, resulting from inefficient nuclear delivery of the DNA (Gupta et al., 2005). 3.1. The importance of drug delivery to the nucleus In the case of drug delivery, the nucleus is also one of the most sensitive sites for drug-induced damage, such as in the case of photosensitising agents used in photodynamic therapy. Photodynamic therapy relies upon the specific activation of photosensitisors such as protoporphoryn IX or chlorin e6 using long wave-length light, releasing singlet oxygen species, the cytotoxic effects of which do not usually exceed 40 nm from the site of activation (Sobolev et al., 2000). Since the nucleus is a hypersensitive site for active oxygen species-induced damage, coupling of the optimised T-ag NLS to chlorin e6-containing complexes, either cross-linked to a carrier or encoded as part of a fusion protein can result in a 2000-fold reduction of the EC50, highlighting the importance of specific nuclear delivery of these chemical agents (Akhlynina et al., 1997, 1999; Rosenkranz et al., 2003). In terms of cancer therapy, delivery of these active agents specifically to the nucleus of tumour, but not normal cells, is the key to their being “magic bullets” in terms of effective and safe anti-tumour therapeutics. 3.2. Tumour cell-specific nuclear targeting Several proteins have been demonstrated to kill tumour cells selectively, including the tumour necrosis factor-related apoptosis inducing ligand (TRAIL) (Walczak et al., 1999), the human αlactalbumin made lethal to tumour cells (HAMLET) (Gustafsson et al., 2005), the adenovirus type 2 early region 4 open reading frame 4 (E4orf4) (Lavoie et al., 1998) and the chicken anemia virus viral protein 3 (Backendorf et al., 2008; Maddika et al., 2006). TRAIL specifically kills malignant cells, but importantly, is able to spare normal tissues, by binding to death receptors expressed exclusively on cancer cells and triggering apoptosis through a mitochondriaindependent signalling pathway (Walczak et al., 1999). Similarly, nuclear localisation appears to be important for the efficiency of killing, but is not essential for E4orf4 induction of p53-independent apoptosis (Lavoie et al., 1998). In a similar fashion to TRAIL, HAMLET induces apoptosis in tumour cells, but leaves normal differentiated cells unharmed (Gustafsson et al., 2005). HAMLET has been shown to kill a wide range of cells in vitro (Gustafsson et al., 2005), to be effective in patients with skin papillomas (Gustafsson et al., 2004), and has been demonstrated to diffuse throughout the brain of a rat xeonograft glioblastoma model, killing tumour cells selectively and halting tumour progression, with little to no apparent normal tissue toxicity (Fischer et al., 2004). Likewise, chicken anemia virus viral protein 3, also known as Apoptin, is an intriguing case, because it is believed to induce tumour cell-selective cell killing, specifically dependent on its ability to localise in the nucleus in tumour, but not normal cells (Alvisi et al., 2006; Backendorf et al., 2008; Maddika et al., 2006). Apoptin is a 121-amino acid protein, which can induce apoptosis in chicken thymocytes and several human tumour but not normal cells, through what appears to be a p53-independent, Bcl-2 resistant pathway (Danen-Van Oorschot et al., 1997; Noteborn, 2005; Tavassoli et al., 2005; Zhuang et al., 1995b). It has also been reported that both primary human hepatocytes and human bone marrow-derived mesenchymal stem cells, which are often destroyed during conventional chemotherapeutic approaches leading to deleterious side effects, are not susceptible to Apoptin-induced apoptosis (Zhang et al., 2003). However, the use of non-isogenic cell pairs in several studies (Danen-Van Oorschot et al., 1997, 2003; Zhuang et al., 1995a,b), combined with recent reports demonstrating programmed cell death in non-transformed primary human embryonal fibroblasts (Guelen et al., 2004) suggests that Apoptin-induced apoptosis may not be specific to tumour cells. Nonetheless, where

177

isogenic cell pairs have been examined, such as the human osteosarcoma cell line SAOS-2 and its normalised counterpart SR40 (SAOS-2 cells stably transfected to express full length Rb (Huang et al., 1988) cells, Apoptin shows enhanced nuclear accumulation in the tumour/ transformed line (Kuusisto et al., 2008; Oro and Jans, 2004; Poon et al., 2005b). Thus, regardless of Apoptin's tumour cell-killing capability, its preferential localisation in the nucleus of tumour/transformed cells (Heilman et al., 2006; Kuusisto et al., 2008; Poon et al., 2005a,b; Wadia et al., 2004; Wang et al., 2004) makes it an intriguing possibility as a targeting moiety for modular cancer therapy applications. 3.2.1. The C-terminus of Apoptin represents a tumour cell-enhanced nuclear targeting signal As noted above, it has been well established that Apoptin localises preferentially in the nucleus of tumour/transformed cells, although it is not an all or nothing situation, whereby some nuclear protein is still present in normal cells, albeit to a lesser extent (Heilman et al., 2006; Lee et al., 2007; Poon et al., 2005a,b). Apoptin contains a basic bipartite NLS in its C-terminus (residues 80–121; KPPSKKRSCDPSEYRVSKLKESLITTTPSRPRTAKRRRIKL121), which is necessary and sufficient for nuclear accumulation (Danen-Van Oorschot et al., 2003; Heilman et al., 2006; Poon et al., 2005a,b). Point mutations in either stretch of basic amino acids impedes nuclear import (Danen-Van Oorschot et al., 2003; Wang et al., 2004), whilst fusion of this motif to heterologous proteins such as green fluorescent protein or maltose-binding protein will confer nuclear localisation (Poon et al., 2005a,b; Rohn et al., 2005; Wadia et al., 2004). Interestingly, we have demonstrated that Apoptin is specifically recognised by Importin β and that when the N-terminus of Apoptin is removed, Apoptin (74–121) binds to Importin β with 3-fold higher affinity, implying that Apoptin's nuclear accumulation may be regulated by intramolecular masking of the NLS through the N-terminus (Wagstaff and Jans, 2006a). Apoptin also possesses a CRM-1 recognised NES (amino acids 97–105; VSKLKESLI105) located between the two basic clusters of the bipartite NLS, which appears to be the basis for tumour cell-enhanced nuclear localisation of Apoptin (Poon et al., 2005a,b). Due to the presence of both NLS and NES sequences, Apoptin has been shown to shuttle between the nucleus and the cytoplasm (Heilman et al., 2006; Poon et al., 2005b). Most importantly, the NES appears to be regulated by phosphorylation at residue T108 in tumour but not normal cells, whereby phosphorylation of this residue in tumour cells inhibits NES-mediated nuclear export of Apoptin, resulting in increased nuclear accumulation (Rohn et al., 2002). This was confirmed formally in a study by Poon et al., (2005b) (see also (Kuusisto et al., 2008)), who examined the localisation of GFP-Apoptin in isogenic tumour/normal cell pairs, demonstrating that treatment with the specific CRM-1 inhibitor Leptomycin B resulted in increased nuclear accumulation only in tumour cells, demonstrating that active nuclear export of Apoptin only occurs in normal but not tumour cells (Poon et al., 2005a). Thus, residues 74–121 encompassing both the NLS(s) and the phosphorylation regulated NES represent a tumour cellenhanced nuclear targeting signal (tNTS), responsible and sufficient for Apoptin's increased nuclear localisation in tumour cells (Kuusisto et al., 2008; Poon et al., 2005b). Clearly the Apoptin tNTS represents an exciting prospect in a therapeutic context to deliver drugs or DNA specifically to the nucleus of tumour cells, thereby increasing both the safety and efficacy of anti-tumour therapies. Interestingly, Apoptin may also prove useful in the diagnosis of cancers, due to its ability to act as an early sensor of carcinogenic transformation, based on the ability of tumour cell lysates to specifically phosphorylate Apoptin at T108 (Backendorf et al., 2008; Kooistra et al., 2007; Maddika et al., 2006; Noteborn, 2005; Russo et al., 2006). 4. A new class of modular transporters with multifactorial targeting systems: The tNTS at the forefront? The idea that chemotherapeutics will reach a point where tumours can be cured by a single ‘magic bullet’ treatment that kills malignant

178

K.M. Wagstaff, D.A. Jans / European Journal of Pharmacology 625 (2009) 174–180

cells and leaves all normal cells untouched may be naïve. It is more likely that a combinatorial approach, using different targeting moieties to confer more specific and potent levels of tumour cell targeting/killing will be successful (see Fig. 3). The fact that it confers preferential accumulation in the nucleus of tumour as opposed to normal cells makes the Apoptin tNTS (see above) a very attractive possibility for use in such a modular therapeutic system. However, since Apoptin can also accumulate in the nucleus of normal cells, albeit to a lesser extent than their transformed counterparts (Kuusisto et al., 2008; Poon et al., 2005a), it seems clear that a multifaceted approach will be required to ensure specific killing of tumour cells without damage to normal cells. Utilisation of several moieties, each having tumour cell-enhanced properties in a novel modular system should result in an overall tumour cell-specific therapeutic drug delivery system with amplified tumour cell targeting/killing properties (Alvisi et al., 2006). This new class of combinatorial cancer therapeutic agents will involve several layers of tumour cell targeting (Fig. 3). The initial step will be to utilise general tumour-targeting approaches to increase the concentration of the drug/DNA/photosensitising agent at or near the intended target cells. This may include administration at or near the target site via direct injection for solid tumours or inhalation for lung epithelial cells, as well as taking advantage of the increased vascularisation common to tumours and the concurrent ‘enhanced permeability and retention effect’ to increase the uptake into tumour tissues (Greish, 2007). Photosensitising agents are an excellent example of this phenomenon, whereby they accumulate preferentially in tumours due to the enhanced tumour vascular permeability (Greish, 2007). However, despite some efficacy of the agents when localised specifically at the cell membrane, internalisation and in particular nuclear targeting results in N2000-fold greater phototoxicity (Gilyazova et al., 2006; Rosenkranz et al., 2003). It should also be stressed that physical/mechanical means to target tumours, although feasible in the case of solid tumours, are not suited to other types of

Fig. 3. Tumour cell-specific strategies. Many levels of tumour cell-specificity are required for drug delivery, depicted schematically from the most general (bottom) to the more specific (top). Each level can be utilised in combination with those below to accomplish a greater efficiency coupled with specificity of tumour targeting. Whilst the more general approaches are suitable for solid tumours, they are not appropriate for leukemic cells/metastases etc., meaning that the other levels of targeting are essential. Ultimately, it is likely that most, if not all of these strategies will need to be employed to achieve the ultimate aim of an effective and above all specific anti-cancer therapeutic (‘magic bullet’).

cancer such as leukaemia or metastatic tumours, wherein novel alternative approaches to take the drug/DNA to the target sites/cells directly are required. Beyond this basic level of targeting, modular recombinant transporters may be engineered to include ligands that are recognised by cellsurface receptors that are either enriched or specific to tumour cells (Gilyazova et al., 2006; Rosenkranz et al., 2003; Sobolev, 2008). For example, receptor tyrosine kinases are frequently overexpressed during cancer development and are often specific to tumour types; e.g., overexpression of Her-2 is associated with breast cancers (Guillemard and Saragovi, 2004). Receptor tyrosine kinases can be targeted using single chain ligands, either in the form of antibody fragments or as short high-affinity peptides; if these are utilised as a cell targeting moiety within a modular system, these should represent the first line of selective tumour cell-specificity in a combinatorial tumour cell-killing approach. One such modular transporter that has met with relative success incorporated an α-melanocyte-stimulating hormone as an internalisable cell-specific ligand, an optimised version of the T-ag NLS as a nuclear targeting moiety, a haemoglobin-like protein as the drug carrier, and the diphtheria toxin translocation domain to effect endosomal escape (Akhlynina et al., 1997, 1999; Rosenkranz et al., 2003). This construct delivers photosensitising agents to the nuclei of αmelanocyte-stimulating hormone receptor overexpressing melanoma cells, resulting in cytotoxicity several orders of magnitude above the photosensitising agents alone (Rosenkranz et al., 2003). Further, the αmelanocyte-stimulating hormone moiety has been able to be replaced with epidermal growth factor as an internalisable ligand for ErbB1 receptors, resulting in N3000 times greater efficacy compared to free photosensitiser alone in A431 human epidermoid carcinoma cells expressing high levels of the ErbB1 receptor; importantly, the modular transport complexes were not photocytotoxic to cells expressing low concentrations of the ErbB1 receptor (Gilyazova et al., 2006). A similar, more recent modular transporter replaced the drug carrier module with the DNA-compacting sperm protein protamine, resulting in a complex that self-assembled DNA into compact nanoparticles that are efficiently trafficked to the nucleus even of non-dividing cells, resulting in enhanced gene expression (Glover et al., 2009). However since cancer cell-specificity in these instances is conferred by a single receptor, there is little to no cancer cell-specific amplification of cell killing. By incorporating more than one cancer cell-specific ligand into the system, such as utilising two or more modular ligands for receptor tyrosine kinases, such as ligands for Her2 and the related epidermal growth factor receptor, both of which can be overexpressed in multiple tumours (Guillemard and Saragovi, 2004), may lead to an amplification of the tumour cell-selective effect, resulting in increased uptake of the drug/ DNA in tumour compared to normal cells. Combining these tumour-selective targeting moieties with novel intracellular targeting modules should amplify the cancer-specific activity of these compounds and may also help to overcome drug efflux problems (Turner and Sullivan, 2008) associated with multidrug resistance proteins such as the ATP-binding cassette transporter ABCG2, which is overexpressed in breast cancer cells (Cusatis and Sparreboom, 2008; Robey et al., 2009). The primary targeting modules to be included here are novel tumour cell-enhanced nuclear targeting signals such as the Apoptin tNTS, which will deliver the photosensitising drug or DNA directly to the hypersensitive nucleus within tumour cells, thus amplifying tumour cell-specific action/cell killing. Alternatively, or perhaps in combination, so called DNA nuclear targeting sequences (DTS) may also be developed/utilised (Dean et al., 1999; Miller and Dean, 2008; Vacik et al., 1999). DTSs are specific DNA sequences that can be incorporated into therapeutic/ plasmid DNA and are recognised by transcription factors/nuclear proteins present in specific cell types, resulting in nuclear uptake of the DNA only in those cells. DTSs discovered so far include the SV40 enhancer region (Dean et al., 1999; Vacik et al., 1999) and the smooth muscle γ-actin promoter region, which promote nuclear uptake of

K.M. Wagstaff, D.A. Jans / European Journal of Pharmacology 625 (2009) 174–180

DNA only in smooth muscle cells and not surrounding tissues (Miller and Dean, 2008). Although cancer specificity of any of these sequences has not yet been described, upregulation of various transcription factors in numerous tumours implies that tumourspecific DTS signals should be able to be developed in similar fashion to the afore-mentioned cell-surface molecules of tumour cells. Additional modules that may act to enhance the intracellular trafficking of the complexes are also desirable. Of interest in this regard may be microtubule association sequences, which mediate interaction with microtubules directly or with components of the motor proteins dynein or kinesin, which mediate transport along microtubules towards the nucleus or cell periphery respectively (Moseley et al., 2007; Roth et al., 2007). Several known viral and cancer-related proteins, such as p53, Rb and rabies virus P-protein contain microtubule association sequences that exploit dyneinmediated movement towards the nucleus along microtubules, resulting in increased and faster nuclear accumulation of NLScontaining proteins (Roth et al., 2007; Moseley, et al., in preparation). Incorporation of such a moiety should help a modular recombinant transporter navigate efficiently through the crowded cytosol towards the nucleus, thus enhancing the nuclear delivery of the gene or drug of interest. In addition, it has been shown that oestrogen treatment of MCF-7 human breast cancer cells elevates dynein light chain expression (Rayala et al., 2005), raising the possibility that upregulation of dynein light chains in tumour cells may further contribute to tumour cell-specificity of recombinant transporters containing microtubule association sequences. Finally, by combining these multi-level combinatorial tumourspecific drug/DNA delivery agents with tumour cell-enhanced killing techniques including utilising tumour cell-specific promoters to generate toxic gene products (for suicide gene therapy applications) or tumour directed light sources (for photodynamic therapy), a multifaceted approach to truly specific anti-tumour therapy, resembling the much anticipated, fabled ‘magic bullet’ should be achieved (Fig. 3). 5. Towards the future The modular recombinant transporters described here reflect a conceptual switch in anti-cancer therapy, from mono-therapy involving a single anti-tumour target/drug, to a combinatorial approach (Alvisi et al., 2006). Through the use of these agents we should, in future be able to kill tumour cells effectively without damaging nearby healthy bystander cells. This will result in less damage to the patient and a much better outcome after treatment. Much remains to be done, but it would seem that achieving the ultimate goal of safe, effective and above all cell-type specific new therapies for the treatment of some of the world's most life-threatening cancers is fast becoming a feasible if not realistic possibility. Importantly, this seems likely to drive the pharmaceutical industry much more in the direction of efficient targeting of existing drugs to particular cell types, and away from drug discovery. References Akhlynina, T.V., Jans, D.A., Rosenkranz, A.A., Statsyuk, N.V., Balashova, I.Y., Toth, G., Pavo, I., Rubin, A.B., Sobolev, A.S., 1997. Nuclear targeting of chlorin e6 enhances its photosensitizing activity. J. Biol. Chem. 272, 20328–20331. Akhlynina, T.V., Jans, D.A., Statsyuk, N.V., Balashova, I.Y., Toth, G., Pavo, I., Rosenkranz, A.A., Naroditsky, B.S., Sobolev, A.S., 1999. Adenoviruses synergize with nuclear localization signals to enhance nuclear delivery and photodynamic action of internalizable conjugates containing chlorin e6. Int. J. Cancer 81, 734–740. Alvisi, G., Jans, D.A., Guo, J., Pinna, L.A., Ripalti, A., 2005. A protein kinase CK2 site flanking the nuclear targeting signal enhances nuclear transport of human cytomegalovirus ppUL44. Traffic 6, 1002–1013. Alvisi, G., Poon, I.K., Jans, D.A., 2006. Tumor-specific nuclear targeting: promises for anti-cancer therapy? Drug Resist. Updat. 9, 40–50. Aris, A., Villaverde, A., 2003. Engineering nuclear localization signals in modular protein vehicles for gene therapy. Biochem. Biophys. Res. Commun. 304, 625–631. Azuma, Y., Dasso, M., 2000. The role of Ran in nuclear function. Curr. Opin. Cell Biol. 12, 302–307.

179

Backendorf, C., Visser, A.E., de Boer, A.G., Zimmerman, R., Visser, M., Voskamp, P., Zhang, Y.H., Noteborn, M., 2008. Apoptin: therapeutic potential of an early sensor of carcinogenic transformation. Annu. Rev. Pharmacol. Toxicol. 48, 143–169. Bayliss, R., Corbett, A.H., Stewart, M., 2000a. The molecular mechanism of transport of macromolecules through nuclear pore complexes. Traffic 1, 448–456. Bayliss, R., Littlewood, T., Stewart, M., 2000b. Structural basis for the interaction between FxFG nucleoporin repeats and importin-beta in nuclear trafficking. Cell 102, 99–108. Box, M., Parks, D.A., Knight, A., Hale, C., Fishman, P.S., Fairweather, N.F., 2003. A multidomain protein system based on the HC fragment of tetanus toxin for targeting DNA to neuronal cells. J. Drug Target. 11, 333–343. Cho, K.C., Kim, S.H., Jeong, J.H., Park, T.G., 2005. Folate receptor-mediated gene delivery using folate–poly(ethylene glycol)–poly(L-lysine) conjugate. Macromol. Biosci. 5, 512–519. Conner, S.D., Schmid, S.L., 2003. Regulated portals of entry into the cell. Nature 422, 37–44. Cusatis, G., Sparreboom, A., 2008. Pharmacogenomic importance of ABCG2. Pharmacogenomics 9, 1005–1009. Danen-Van Oorschot, A.A., Fischer, D.F., Grimbergen, J.M., Klein, B., Zhuang, S., Falkenburg, J.H., Backendorf, C., Quax, P.H., Van der Eb, A.J., Noteborn, M.H., 1997. Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells. Proc. Natl. Acad. Sci. U. S. A. 94, 5843–5847. Danen-Van Oorschot, A.A., Zhang, Y.H., Leliveld, S.R., Rohn, J.L., Seelen, M.C., Bolk, M.W., Van Zon, A., Erkeland, S.J., Abrahams, J.P., Mumberg, D., Noteborn, M.H., 2003. Importance of nuclear localization of apoptin for tumor-specific induction of apoptosis. J. Biol. Chem. 278, 27729–27736. Dean, D.A., Dean, B.S., Muller, S., Smith, L.C., 1999. Sequence requirements for plasmid nuclear import. Exp. Cell Res. 253, 713–722. Escriou, V., Carriere, M., Scherman, D., Wils, P., 2003. NLS bioconjugates for targeting therapeutic genes to the nucleus. Adv. Drug Deliv. Rev. 55, 295–306. Fischer, W., Gustafsson, L., Mossberg, A.K., Gronli, J., Mork, S., Bjerkvig, R., Svanborg, C., 2004. Human alpha-lactalbumin made lethal to tumor cells (HAMLET) kills human glioblastoma cells in brain xenografts by an apoptosis-like mechanism and prolongs survival. Cancer Res. 64, 2105–2112. Fukuda, M., Asano, S., Nakamura, T., Adachi, M., Yoshida, M., Yanagida, M., Nishida, E., 1997. CRM1 is responsible for intracellular transport mediated by the nuclear export signal. Nature 390, 308–311. Gilyazova, D.G., Rosenkranz, A.A., Gulak, P.V., Lunin, V.G., Sergienko, O.V., Khramtsov, Y.V., Timofeyev, K.N., Grin, M.A., Mironov, A.F., Rubin, A.B., Georgiev, G.P., Sobolev, A.S., 2006. Targeting cancer cells by novel engineered modular transporters. Cancer Res. 66, 10534–10540. Glover, D.J., Lipps, H.J., Jans, D.A., 2005. Towards safe, non-viral therapeutic gene expression in humans. Nat. Rev. Genet. 6, 299–310. Glover, D.J., Ng, S.M., Mechler, A., Martin, L.L., Jans, D.A., 2009. Multifunctional protein nanocarriers for targeted nuclear gene delivery in nondividing cells. FASEB J. 23 (9), 2996–3006. Goldfarb, D.S., Corbett, A.H., Mason, D.A., Harreman, M.T., Adam, S.A., 2004. Importin alpha: a multipurpose nuclear-transport receptor. Trends Cell Biol. 14, 505–514. Gorlich, D., 1998. Transport into and out of the cell nucleus. EMBO J. 17, 2721–2727. Gorlich, D., Kutay, U., 1999. Transport between the cell nucleus and the cytoplasm. Annu. Rev. Cell Dev. 15, 607–660. Greish, K., 2007. Enhanced permeability and retention of macromolecular drugs in solid tumors: a royal gate for targeted anticancer nanomedicines. J. Drug Target. 15, 457–464. Guelen, L., Paterson, H., Gaken, J., Meyers, M., Farzaneh, F., Tavassoli, M., 2004. TATapoptin is efficiently delivered and induces apoptosis in cancer cells. Oncogene 23, 1153–1165. Guillemard, V., Saragovi, H.U., 2004. Novel approaches for targeted cancer therapy. Curr. Cancer Drug Targets 4, 313–326. Gupta, B., Levchenko, T.S., Torchilin, V.P., 2005. Intracellular delivery of large molecules and small particles by cell-penetrating proteins and peptides. Adv. Drug Deliv. Rev. 57, 637–651. Gustafsson, L., Leijonhufvud, I., Aronsson, A., Mossberg, A.K., Svanborg, C., 2004. Treatment of skin papillomas with topical alpha-lactalbumin–oleic acid.[see comment]. N. Engl. J. Med. 350, 2663–2672. Gustafsson, L., Hallgren, O., Mossberg, A.K., Pettersson, J., Fischer, W., Aronsson, A., Svanborg, C., 2005. HAMLET kills tumor cells by apoptosis: structure, cellular mechanisms, and therapy. J. Nutr. 135, 1299–1303. Harel, A., Forbes, D.J., 2004. Importin beta: conducting a much larger cellular symphony. Mol. Cell 16, 319–330. Heilman, D.W., Teodoro, J.G., Green, M.R., 2006. Apoptin nucleocytoplasmic shuttling is required for cell type-specific localization, apoptosis, and recruitment of the anaphase-promoting complex/cyclosome to PML bodies. J. Virol. 80, 7535–7545. Hogarth, C.A., Calanni, S., Jans, D.A., Loveland, K.L., 2006. Importin alpha mRNAs have distinct expression profiles during spermatogenesis. Dev. Dyn. 235, 253–262. Huang, H.J., Yee, J.K., Shew, J.Y., Chen, P.L., Bookstein, R., Friedmann, T., Lee, E.Y., Lee, W.H., 1988. Suppression of the neoplastic phenotype by replacement of the RB gene in human cancer cells. Science 242, 1563–1566. Hubner, S., Xiao, C.Y., Jans, D.A., 1997. The protein kinase CK2 site (Ser111/112) enhances recognition of the simian virus 40 large T-antigen nuclear localization sequence by importin. J. Biol. Chem. 272, 17191–17195. Jakel, S., Albig, W., Kutay, U., Bischoff, F.R., Schwamborn, K., Doenecke, D., Gorlich, D., 1999. The importin beta/importin 7 heterodimer is a functional nuclear import receptor for histone H1. EMBO J. 18, 2411–2423. Jans, D.A., Chan, C.K., Huebner, S., 1998. Signals mediating nuclear targeting and their regulation: application in drug delivery. Med. Res. Rev. 18, 189–223. Jans, D.A., Xiao, C.Y., Lam, M.H., 2000. Nuclear targeting signal recognition: a key control point in nuclear transport? Bioessays 22, 532–544.

180

K.M. Wagstaff, D.A. Jans / European Journal of Pharmacology 625 (2009) 174–180

Kaffman, A., Rank, N.M., O'Shea, E.K., 1998. Phosphorylation regulates association of the transcription factor Pho4 with its import receptor Pse1/Kap121. Genes Dev. 12, 2673–2683. Kalderon, D., Richardson, W.D., Markham, A.F., Smith, A.E., 1984a. Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature 311, 33–38. Kalderon, D., Roberts, B.L., Richardson, W.D., Smith, A.E., 1984b. A short amino acid sequence able to specify nuclear location. Cell 39, 499–509. Keller, M., Harbottle, R.P., Perouzel, E., Colin, M., Shah, I., Rahim, A., Vaysse, L., Bergau, A., Moritz, S., Brahimi-Horn, C., Coutelle, C., Miller, A.D., 2003. Nuclear localisation sequence templated nonviral gene delivery vectors: investigation of intracellular trafficking events of LMD and LD vector systems. Chembiochem 4, 286–298. Khalil, I.A., Kogure, K., Akita, H., Harashima, H., 2006. Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery. Pharmacol. Rev. 58, 32–45. Komeili, A., O'Shea, E.K., 1999. Roles of phosphorylation sites in regulating activity of the transcription factor Pho4. Science 284, 977–980. Kooistra, K., Zhang, Y.H., Noteborn, M.H., 2007. Viral elements sense tumorigenic processes: approaching selective cancer therapy. Mini-Rev. Med. Chem. 7, 1155–1165. Kudo, N., Wolff, B., Sekimoto, T., Schreiner, E.P., Yoneda, Y., Yanagida, M., Horinouchi, S., Yoshida, M., 1998. Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1. Exp. Cell Res. 242, 540–547. Kuusisto, H.V., Wagstaff, K.M., Alvisi, G., Jans, D.A., 2008. The C-terminus of apoptin represents a unique tumor cell-enhanced nuclear targeting module. Int. J. Cancer 123, 2965–2969. Lam, M.H., Briggs, L.J., Hu, W., Martin, T.J., Gillespie, M.T., Jans, D.A., 1999. Importin beta recognizes parathyroid hormone-related protein with high affinity and mediates its nuclear import in the absence of importin alpha. J. Biol. Chem. 274, 7391–7398. Langer, T., 2000. Nuclear transport of histone 2b in mammalian cells is signal- and energy-dependent and different from the importin alpha/beta-mediated process. Histochem. Cell Biol. 113, 455–465. Lavoie, J.N., Nguyen, M., Marcellus, R.C., Branton, P.E., Shore, G.C., 1998. E4orf4, a novel adenovirus death factor that induces p53-independent apoptosis by a pathway that is not inhibited by zVAD-fmk. J. Cell Biol. 140, 637–645. Lee, Y.H., Cheng, C.M., Chang, Y.F., Wang, T.Y., Yuo, C.Y., 2007. Apoptin T108 phosphorylation is not required for its tumor-specific nuclear localization but partially affects its apoptotic activity. Biochem. Biophys. Res. Commun. 354, 391–395. Liu, C.S., Kong, B., Xia, H.H., Ellem, K.A., Wei, M.Q., 2001. VP22 enhanced intercellular trafficking of HSV thymidine kinase reduced the level of ganciclovir needed to cause suicide cell death. J. Gene Med. 3, 145–152. Maddika, S., Mendoza, F.J., Hauff, K., Zamzow, C.R., Paranjothy, T., Los, M., 2006. Cancerselective therapy of the future: apoptin and its mechanism of action. Cancer Biol. Ther. 5, 10–19. Miller, A.M., Dean, D.A., 2008. Cell-specific nuclear import of plasmid DNA in smooth muscle requires tissue-specific transcription factors and DNA sequences. Gene Ther. 15, 1107–1115. Mosammaparast, N., Pemberton, L.F., 2004. Karyopherins: from nuclear-transport mediators to nuclear-function regulators. Trends Cell Biol. 14, 547–556. Mosammaparast, N., Guo, Y., Shabanowitz, J., Hunt, D.F., Pemberton, L.F., 2002. Pathways mediating the nuclear import of histones H3 and H4 in yeast. J. Biol. Chem. 277, 862–868. Moseley, G.W., Roth, D.M., DeJesus, M.A., Leyton, D.L., Filmer, R.P., Pouton, C.W., Jans, D.A., 2007. Dynein light chain association sequences can facilitate nuclear protein import. Mol. Biol. Cell 18, 3204–3213. Nishikawa, M., Yamauchi, M., Morimoto, K., Ishida, E., Takakura, Y., Hashida, M., 2000. Hepatocyte-targeted in vivo gene expression by intravenous injection of plasmid DNA complexed with synthetic multi-functional gene delivery system. Gene Ther. 7, 548–555. Noteborn, M.H., 2005. Apoptin acts as a tumor-specific killer: potentials for an antitumor therapy. Cell. Mol. Biol. 51, 49–60. Oro, C., Jans, D.A., 2004. The tumour specific pro-apoptotic factor apoptin (Vp3) from chicken anaemia virus. Curr. Drug Targets 5, 179–190. Pollard, V.W., Michael, W.M., Nakielny, S., Siomi, M.C., Wang, F., Dreyfuss, G., 1996. A novel receptor-mediated nuclear protein import pathway. Cell 86, 985–994. Poon, I.K., Jans, D.A., 2005. Regulation of nuclear transport: central role in development and transformation? Traffic 6, 173–186. Poon, I.K., Oro, C., Dias, M.M., Zhang, J., Jans, D.A., 2005a. Apoptin nuclear accumulation is modulated by a CRM1-recognized nuclear export signal that is active in normal but not in tumor cells. Cancer Res. 65, 7059–7064. Poon, I.K., Oro, C., Dias, M.M., Zhang, J.P., Jans, D.A., 2005b. A tumor cell-specific nuclear targeting signal within chicken anemia virus VP3/apoptin.[comment]. J. Virol. 79, 1339–1341. Rayala, S.K., den Hollander, P., Balasenthil, S., Yang, Z., Broaddus, R.R., Kumar, R., 2005. Functional regulation of oestrogen receptor pathway by the dynein light chain 1. EMBO Rep. 6, 538–544 [erratum appears in EMBO Rep. 2005 Nov;6(11):1101]. Ribbeck, K., Gorlich, D., 2001. Kinetic analysis of translocation through nuclear pore complexes. EMBO J. 20, 1320–1330. Robbins, P.D., Ghivizzani, S.C., 1998. Viral vectors for gene therapy. Pharmacol. Ther. 80, 35–47. Robbins, J., Dilworth, S.M., Laskey, R.A., Dingwall, C., 1991. Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell 64, 615–623. Robey, R.W., To, K.K., Polgar, O., Dohse, M., Fetsch, P., Dean, M., Bates, S.E., 2009. ABCG2: a perspective. Adv. Drug Deliv. Rev. 61, 3–13. Rohn, J.L., Zhang, Y.H., Aalbers, R.I., Otto, N., Den Hertog, J., Henriquez, N.V., Van De Velde, C.J., Kuppen, P.J., Mumberg, D., Donner, P., Noteborn, M.H., 2002. A tumor-

specific kinase activity regulates the viral death protein Apoptin. J. Biol. Chem. 277, 50820–50827. Rohn, J.L., Zhang, Y.H., Leliveld, S.R., Danen-van Oorschot, A.A., Henriquez, N.V., Abrahams, J.P., Noteborn, M.H., 2005. Relevance of apoptin's integrity for its functional behavior.[comment]. J. Virol. 79, 1337–1338. Rosenkranz, A.A., Lunin, V.G., Gulak, P.V., Sergienko, O.V., Shumiantseva, M.A., Voronina, O.L., Gilyazova, D.G., John, A.P., Kofner, A.A., Mironov, A.F., Jans, D.A., Sobolev, A.S., 2003. Recombinant modular transporters for cell-specific nuclear delivery of locally acting drugs enhance photosensitizer activity. FASEB J. 17, 1121–1123. Roth, D.M., Moseley, G.W., Glover, D., Pouton, C.W., Jans, D.A., 2007. A microtubulefacilitated nuclear import pathway for cancer regulatory proteins. Traffic 8, 673–686. Russo, A., Terrasi, M., Agnese, V., Santini, D., Bazan, V., 2006. Apoptosis: a relevant tool for anticancer therapy. Ann. Oncol. 17 (Suppl 7), vii115–vii123. Seth, P., 1994. Mechanism of adenovirus-mediated endosome lysis: role of the intact adenovirus capsid structure. Biochem. Biophys. Res. Commun. 205, 1318–1324. Sobolev, A.S., 2008. Modular transporters for subcellular cell-specific targeting of antitumor drugs. Bioessays 30, 278–287. Sobolev, A.S., Jans, D.A., Rosenkranz, A.A., 2000. Targeted intracellular delivery of photosensitizers. Prog. Biophys. Mol. Biol. 73, 51–90. Tasciotti, E., Zoppe, M., Giacca, M., 2003. Transcellular transfer of active HSV-1 thymidine kinase mediated by an 11-amino-acid peptide from HIV-1 Tat. Cancer Gene Ther. 10, 64–74. Tavassoli, M., Guelen, L., Luxon, B.A., Gaken, J., 2005. Apoptin: specific killer of tumor cells? Apoptosis 10, 717–724. Turner, J.G., Sullivan, D.M., 2008. CRM1-mediated nuclear export of proteins and drug resistance in cancer. Curr. Med. Chem. 15, 2648–2655. Vacik, J., Dean, B.S., Zimmer, W.E., Dean, D.A., 1999. Cell-specific nuclear import of plasmid DNA. Gene Ther. 6, 1006–1014. Vaughan, E.E., DeGiulio, J.V., Dean, D.A., 2006. Intracellular trafficking of plasmids for gene therapy: mechanisms of cytoplasmic movement and nuclear import. Curr. Gene Ther. 6, 671–681. Wadia, J.S., Wagner, M.V., Ezhevsky, S.A., Dowdy, S.F., 2004. Apoptin/VP3 contains a concentration-dependent nuclear localization signal (NLS), not a tumorigenic selective NLS. J. Virol. 78, 6077–6078. Wagner, E., Cotten, M., Foisner, R., Birnstiel, M.L., 1991. Transferrin-polycation–DNA complexes: the effect of polycations on the structure of the complex and DNA delivery to cells. Proc. Natl. Acad. Sci. U. S. A. 88, 4255–4259. Wagner, E., Zatloukal, K., Cotten, M., Kirlappos, H., Mechtler, K., Curiel, D.T., Birnstiel, M.L., 1992. Coupling of adenovirus to transferrin-polylysine/DNA complexes greatly enhances receptor-mediated gene delivery and expression of transfected genes. Proc. Natl. Acad. Sci. U. S. A. 89, 6099–6103. Wagstaff, K.M., Jans, D.A., 2006a. Intramolecular masking of nuclear localization signals: analysis of importin binding using a novel AlphaScreen-based method. Anal. Biochem. 348, 49–56. Wagstaff, K.M., Jans, D.A., 2006b. Protein transduction: cell penetrating peptides and their therapeutic applications. Curr. Med. Chem. 13, 1371–1387. Wagstaff, K.M., Jans, D.A., 2007. Nucleocytoplasmic transport of DNA: enhancing nonviral gene transfer. Biochem. J. 406, 185–202. Wagstaff, K.M., Glover, D.J., Tremethick, D.J., Jans, D.A., 2007. Histone-mediated transduction as an efficient means for gene delivery. Mol. Ther. 15, 721–731. Wagstaff, K.M., Fan, J.Y., De Jesus, M.A., Tremethick, D.J., Jans, D.A., 2008. Efficient gene delivery using reconstituted chromatin enhanced for nuclear targeting. FASEB J. 22, 2232–2242. Walczak, H., Miller, R.E., Ariail, K., Gliniak, B., Griffith, T.S., Kubin, M., Chin, W., Jones, J., Woodward, A., Le, T., Smith, C., Smolak, P., Goodwin, R.G., Rauch, C.T., Schuh, J.C., Lynch, D.H., 1999. Tumoricidal activity of tumor necrosis factor-related apoptosisinducing ligand in vivo. Nat. Med. 5, 157–163. Wang, Q.M., Fan, G.C., Chen, J.Z., Chen, H.P., He, F.C., 2004. A putative NES mediates cytoplasmic localization of Apoptin in normal cells. Acta Biochim. Biophys. Sin. 36, 817–823. Weir, H.K., Thun, M.J., Hankey, B.F., Ries, L.A., Howe, H.L., Wingo, P.A., Jemal, A., Ward, E., Anderson, R.N., Edwards, B.K., 2003. Annual report to the nation on the status of cancer, 1975–2000, featuring the uses of surveillance data for cancer prevention and control. [see comment]. J. Natl. Cancer Inst. 95, 1276–1299 [erratum appears in J Natl Cancer Inst. 2003 Nov 5;95(21):1641]. Wente, S.R., 2000. Gatekeepers of the nucleus. Science 288, 1374–1377. Zhang, F., White, R.L., Neufeld, K.L., 2001. Cell density and phosphorylation control the subcellular localization of adenomatous polyposis coli protein. Mol. Cell. Biol. 21, 8143–8156. Zhang, Y.H., Leliveld, S.R., Kooistra, K., Molenaar, C., Rohn, J.L., Tanke, H.J., Abrahams, J.P., Noteborn, M.H., 2003. Recombinant Apoptin multimers kill tumor cells but are nontoxic and epitope-shielded in a normal-cell-specific fashion. Exp. Cell Res. 289, 36–46. Zhao, X., Gan, L., Pan, H., Kan, D., Majeski, M., Adam, S.A., Unterman, T.G., 2004. Multiple elements regulate nuclear/cytoplasmic shuttling of FOXO1: characterization of phosphorylation- and 14–3–3-dependent and -independent mechanisms. Biochem. J. 378, 839–849. Zhuang, S.M., Shvarts, A., Jochemsen, A.G., van Oorschot, A.A., van der Eb, A.J., Noteborn, M.H., 1995a. Differential sensitivity to Ad5 E1B–21kD and Bcl-2 proteins of apoptin-induced versus p53-induced apoptosis. Carcinogenesis 16, 2939–2944. Zhuang, S.M., Shvarts, A., van Ormondt, H., Jochemsen, A.G., van der Eb, A.J., Noteborn, M.H., 1995b. Apoptin, a protein derived from chicken anemia virus, induces p53independent apoptosis in human osteosarcoma cells. Cancer Res. 55, 486–489.