Drug Resistance Updates 9 (2006) 40–50

Tumor-specific nuclear targeting: Promises for anti-cancer therapy?

Gualtiero Alvisi a, Ivan K.H. Poon a, David A. Jans a,b,∗a Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Australia

b ARC Centre of Excellence for Biotechnology and Development, Australia

Received 13 January 2006; received in revised form 20 February 2006; accepted 20 February 2006

Abstract

Recent developments in anti-cancer gene therapy suggest that the idea of a magic bullet for cancer may not be a pipe dream. Viral-basedanti-cancer vectors for gene therapy have been used preferentially in this regard, but recent results from clinical trials have raised seriousconcerns as to their safety. For this reason, the development of non-viral vectors able to deliver drugs or suicide genes specifically to cancercells is of paramount importance. In this context, great interest has been raised by recent reports that several proteins, including viral protein3 (VP3 or Apoptin) from Chicken Anemia Virus, are capable of selectively killing tumor cells. Intriguingly, VP3’s anti-cancer activity isstrongly linked to its ability to localize more efficiently in the nucleus of cancer and transformed cells than that of normal, non-transformedcpra©

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ells with a tumor cell-specific nuclear targeting signal (tNTS) located at the C-terminus of the protein. Clearly, the VP3 tNTS is an excitingrospect to enhance non-viral-mediated cancer cell killing. This review will discuss recent advances in the understanding of the mechanismesponsible for VP3 tumor-specific nuclear localization, including its specific phosphorylation, and the implications for the enhancement ofnti-cancer therapy. It also proposes alternative strategies to develop tNTSs for anti-cancer therapies.

2006 Elsevier Ltd. All rights reserved.

eywords: Nuclear targeting; Apoptin; Cancer; Nuclear targeting signal; Apoptosis; Vehicle; Modular transporters; Therapy; Gene therapy; Drug delivery;P3; Anti-cancer; Photosensitizers

. Introduction

Cancer can be regarded as a heterogeneous group of pro-iferative diseases, resulting from the accumulation of geneticesions. Despite considerable advances in our understandingf the molecular mechanisms of carcinogenesis and cancerrogression, cancer remains the second major cause of deathue to medical causes in the United States of America asell as in much of the developed world (Weir et al., 2003).learly, there is an urgent need for new, efficacious and

pecific anti-cancer approaches (Guillemard and Saragovi,004). Virus-based anti-cancer gene therapies, based on thebility of adenoviral or retroviral vectors to deliver suicideenes to cancer cells (Rainov and Ren, 2003), or to kill tumorells directly (“virolytic therapy”; Everts and van der Poel,

∗ Corresponding author at: Nuclear Signalling Laboratory, Department ofiochemistry and Molecular Biology, Monash University, Wellington Road,layton Vic. 3168, Australia.

E-mail address: David.Jans@med.monash.edu.au (D.A. Jans).

2005; Mathis et al., 2005), are being tested in various clini-cal trials (www4.od.nih.gov/oba/rac/protocol.pdf). However,although one viral-based vector has been recently approvedfor the treatment of head and neck cancer, the outcome ofseveral viral-based clinical trials has raised serious concernsregarding the efficacy and safety of such approaches, high-lighting the need to develop alternative strategies (Hacein-Bey-Abina et al., 2003a,b; Raper et al., 2003; Williams andBaum, 2003; Glover et al., 2005; Young et al., 2006). Herewe discuss the possibility of using recently identified tumorcell-specific nuclear targeting sequences (tNTSs) as toolsfor enhancing non-viral drug or suicide gene delivery to thenucleus of cancer cells.

2. Nuclear delivery mechanisms

A useful strategy in anti-cancer therapy is the specificdelivery of high levels of either a suicide gene or a drug(see Box 1) to malignant cells, without affecting healthy

368-7646/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.

oi:10.1016/j.drup.2006.02.003

G. Alvisi et al. / Drug Resistance Updates 9 (2006) 40–50 41

Box 1Herpes simplex virus thymidine kinase asa suicide geneA suicide gene programs cells to kill them-selves under certain circumstances, such asupon administration of an inactive prodrugwhich can be processed to a toxic form. One ofthe most widely used suicide genes is the Her-pes simplex virus 1 (HSV-1) thymidine kinase(TK). HSV-1 TK differs considerably from thehuman cellular TK in its substrate specificity,phosphorylating a broad spectrum of pyrimi-dine and purine nucleoside analogues (Pilgeret al., 1999), including ganciclovir (GCV). HSV-1but not human TK phosphorylates GCV to itsmonophosphate form, which is subsequentlyconverted to the triphosphorylated derivativeby cellular kinases. The active triphosphory-lated metabolite is then incorporated into thenewly synthetized DNA chain, thereby blockingDNA replication. Thus, HSV-1 TK is essentialfor GCV-induced cellular toxicity, and GCV isone of the most widely used antiviral drugs fortreatment of viral infections (De Clercq, 2004).Because of its unique properties, the HSV-1 TKgene has been widely used as a suicide genein gene therapy vectors (Vassaux and Martin-Duque, 2004).Photodynamic therapyPhotodynamic therapy (PDT) is based on theability of photosentitizers (PSs) such as chlo-rin e6, to generate singlet oxygen species inresponse to light exposure. PSs preferentiallyaccumulate in tumor cells but also in some nor-mal tissues such the skin (Jori, 1996), and theiraction is strongly dependent on their localiza-tion inside the target cell, as their cytotoxicaction does not exceed 40 nm from the site ofactivation (Sobolev et al., 2000). To increasetheir specificity, PSs can be directly admin-istrated into the tumoral mass, in combina-tion with direct light exposure (Sharman et al.,1999). PDT has been proven to be effectivefor the treatment of some cancers (Wiedmannet al., 2003), with the nucleus being the mostsensitive subcellular compartment (Peng et al.,1996). For this reason, development of methodsto increase PS targeting to the nucleus of tumorcells is desirable (Bisland et al., 1999; Sobolevet al., 2000).

cells and tissues. Viral-based vehicles represent the bestoption in terms of the efficiency of delivery, as viruses haveevolved very specific mechanisms to deliver genetic materialand proteins to host cells (Fig. 1).

Viruses can interact with the host cell via specific interac-tion of viral proteins with cellular receptors exposed on theextracellular surface of the cellular plasma membrane (PM;see Fig. 1.1). In the case of non-enveloped DNA viruses suchas members of the Adenoviridae family, the virus can be inter-nalized by the cells through an endocytotic mechanism sub-sequent to receptor binding (see Fig. 1.2). Receptor-mediatedendocytosis usually leads to degradation of the internalizedligand in the lysosomal pathway, but certain viral proteinsundergo conformational changes under the acid conditions ofthe endosome to disrupt the vesicular membrane and facili-tate the release of the viral particles into the cytoplasm of thehost cell (see Fig. 1.3; Seth, 1994).

In the case of most DNA viruses and retroviruses, the viralgenome is ultimately translocated into the nucleus of the hostcell (see Fig. 1.4), in order to be expressed and replicated, orintegrated into the host genome in the case of retroviruses.The eukaryotic cell nucleus is separated from the rest of thecell by a double membrane structure – the nuclear envelope(NE) – the only passage through which is represented bythe c. 120 MDa, multiprotein-constituted nuclear pore com-plexes (NPCs). Molecules with a mass of < 50 kDa can freelydwas�olf(taa1(1eirtNisINtrlCir

iffuse through the aqueous channel delimited by the NPC,hereas nucleocytoplasmic transport of larger molecules is

n active process mediated by members of the importin (IMP)uperfamily of soluble cellular transporters, which can be ofand � types (Weis, 1998). Members of the IMP family rec-

gnize specific targeting signals on cargoes, named nuclearocalization signals (NLSs) or nuclear export signals (NESs)or transport in the import and export directions, respectivelyFig. 2). NLSs and NESs were first identified on viral pro-eins: the first NLS characterized, the highly basic 7 aminocid sequence (PKKKRKV132), was from the large tumorntigen (T-Ag) from Simian Virus SV40 (Kalderon et al.,984), whereas a strongly hydrophobic 11 amino acid stretchLQLPPLERLTL83), from the human immunodeficency virus(HIV-1) Rev protein was the first NES described (Meyer

t al., 1996). The best-characterized nuclear import pathways that mediated by the IMP�/� heterodimer, where IMP�ecognizes the NLS (such as that of T-Ag) on the cargo pro-ein, and IMP� mediates the docking of the complex to thePC (see Fig. 2, left; Weis, 1998). IMP� itself, or one of

ts many homologues, is also able to interact directly withpecific cargoes independently of IMP�. Subsequently theMP-cargo complex is translocated to the nuclear side of thePC, where binding of the guanidine nucleotide binding pro-

ein Ran in activated GTP-bound form to IMP� results in theelease of the cargo into the nucleus. Certain IMP� homo-ogues mediate nuclear protein export, the best known beingRM-1 (Fukuda et al., 1997), whose function can be inhib-

ted specifically by the drug leptomycin B (LMB; see Fig. 2,ight; Kudo et al., 1998).

42 G. Alvisi et al. / Drug Resistance Updates 9 (2006) 40–50

Fig. 1. Intracellular delivery as mediated by viruses and modular transporters. Viral particles such as that of adenovirus and modular transporters are able tointeract specifically with a cell surface receptor (1), leading to receptor-mediated endocytosis (2). Certain viral and other proteins can undergo conformationalmodifications triggered by the acidic endosomal pH, causing disruption of the endosome (3) and the release of the viral particle or of the modular transporterinto the cytoplasm so that they can subsequently be imported into the cell nucleus (4), where the genetic material is finally able to be expressed, or the drugreleased (5). PM, plasma membrane; NE, nuclear envelope; CT, cellular targeting, EE, endosomal escape; NT, nuclear targeting.

The three functions described above – cell targeting (CT),endosomal escape (EE), and nuclear targeting (NT; see Fig. 1)– are desirable elements of an efficient cancer-specific target-ing agent (Glover et al., 2005). Modular recombinant trans-porters, assemblies of independently acting domains able tofulfill some or all of these functions (see Table 1 and Fig. 1),have been shown to be capable of targeting reporter genesor drugs specifically to cancer cells in culture (see below;Rosenkranz et al., 2003). That modular transporters of thistype may be suitable for targeted delivery in the clinic inthe future is indicated by the observation that liver-targetedluciferase expression has been achieved in mice after intra-venous injection of a modular transporter complexed withplasmid DNA (Nishikawa et al., 2000).

3. Importance of nuclear delivery of modulartransporters

Several studies have shown that the efficiency of foreigngene expression depends on the ability of the modular trans-porter to deliver the coding sequences to the nucleus of thetarget cells and that addition of NT modules can improve geneexpression (Chan et al., 2000; Chan and Jans, 1999a,b, 2001,2002). For example, Villaverde and coworkers described amodular protein (249AL) comprising the �-galactosidase (�-GDm

able to interact with cell surface integrins. 249AL is able toassociate with a firefly luciferase expressing plasmid withoutundergoing any appreciable diminution of �-Gal enzymaticactivity and can facilitate expression of the luciferase reporterin integrin-expressing CaCO2 cells with an efficiency com-parable to that of a non-targeted liposome-based commercialtransfection system (Aris et al., 2000). Subsequent inclusionof an NT module by adding the T-Ag NLS, generated theNLSCt protein which transfected CaCO2 cells with 30-foldhigher efficiency than that of 249AL. The implication is thatthe efficiency of transgene expression can be facilitated byincreasing the nuclear delivery of the transgene (Aris andVillaverde, 2003).

Intranuclear delivery is also important for cellular trans-porters designed to deliver photosentitizing (PS) agents tocancer cells (see Box 1). Using bovine serum albumin (BSA)as a carrier linked to insulin as a CT module, Sobolev andcoworkers (Akhlynina et al., 1993, 1995) generated a modulartargeting protein able to specifically deliver conjugated clorine6 to the human hepatoma cell line PLC/PRF/5, resulting inreduction of the EC50 by about 100-fold when compared tothat of free clorin e6 after light exposure. Interestingly, addi-tion of a functional “optimized” T-Ag NLS (opT-NLS) as anNT module significantly decreased the EC50 about 7-fold,compared to a functionally impaired NLS mutant derivative(Akhlynina et al., 1997). Finally, addition of an EE moietyit1

al) protein as a carrier, a N-terminal polylysine stretch as aNA compacting and protecting module, and the foot-and-outh disease virus (FMDV) RGD sequence as a CT module

n the form of adenovirus particles mixed with the modularransporter further enhanced the efficiency (Akhlynina et al.,999).

G. Alvisi et al. / Drug Resistance Updates 9 (2006) 40–50 43

Fig. 2. Schematic representation of nucleocytoplasmic transport. (A) Nuclear import occurs when IMP�1, either alone or through IMP�, recognizes an NLSon the cargo protein in the cytoplasm (1) and mediates the docking of the complex to the NPC (2) followed by translocation into the cell nucleus. Binding ofRanGTP to IMP�1 (3) results in the cargo being released into the nucleus (4). (B) Nuclear export occurs when cellular export receptors, such as the IMP�

homologue CRM-1, recognize a NES on the cargo protein in the presence of RanGTP (1) and mediate the docking of the complex to the NPC and its translocationto the cytoplasm (2). Upon hydrolysis by Ran of GTP to GDP (3), facilitated by Ran GTPase-activating protein (not shown), the cargo is released into thecytoplasm (4).

These and other studies show that the addition of anNT module to a modular transporter significantly enhancesthe efficacy of both gene transfer (Chan et al., 1998, 2000;Chan and Jans, 1999a,b, 2001; Singh et al., 1999; Aris andVillaverde, 2003; Ritter et al., 2003) and sensitivity of targetcells to nuclear acting toxic agents such as PSs (Akhlynina etal., 1997, 1999; Bisland et al., 1999; Brokx et al., 2002; seealso Table 1).

4. Tumor cell-specific nuclear delivery

Several proteins, including TRAIL, Adenovirus E4orf4and the Chicken Anemia Virus protein 3 (VP3, also knownas Apoptin) have been shown to kill tumor cells selec-tively (Danen-van Oorschot et al., 1997; Lavoie et al., 1998;

Walczak et al., 1999), raising great hopes for their utiliza-tion in anti-cancer therapy (Branton and Roopchand, 2001;MacFarlane, 2003; Rohn and Noteborn, 2004; Tavassoliet al., 2005). While nuclear localization is important, butnot essential, for E4orf4 mediated induction of apop-tosis (Miron et al., 2004), VP3’s ability to kill tumorcells selectively is correlated with its nuclear localizationspecifically in tumor and not “normal” cells (Danen-vanOorschot et al., 2003). VP3 is a 121-amino acid pro-tein rich in Ser and Thr residues, which induces pro-grammed cell death (PCD) in chicken thymocytes (Notebornet al., 1994), and in several human tumor but not normalcells, through a p53-independent, Bcl-2-resistant process(Zhuang et al., 1995). However, its ability to induce PCDin primary human fibroblasts has been reported recently(Guelen et al., 2004), suggesting that further studies are

44 G. Alvisi et al. / Drug Resistance Updates 9 (2006) 40–50

Table 1

CT EE NT PS agent Reference

A. Modular proteins used to deliver photosensitizing (PS) agents to mammalian cellsa

Insulin – – Chlorin e6 Akhlynina et al. (1995)Insulin – opT-NLS(1); T-ag NLS Chlorin e6 Akhlynina et al. (1997)Insulin Adenoviral particles opT-NLS Chlorin e6 Akhlynina et al. (1999)�-MSH DT TD opT-NLS Chlorin e6 Rosenkranz et al. (2003)

Bacterio chlorin P6EGF – – Aluminium/Cobalt Lutsenko et al. (1999)

Disulfonated phthalocyaninesEGF – – Tin (IV) chlorin e6 Gijsens et al. (2000)Polylysine – A124SV40LTa Chlorin e6 Bisland et al. (1999)Maleic acid – – Chlorin e6 Liu and Hamblin (2005)

CT EE NT DNA binding Reference

B. Modular proteins used to deliver DNA to mammalian cellsb

– – NLSV404 peptide(2) NLSV404 peptide Ritter et al. (2003)Integrin binding domain of Yersinia

pseudotuberculosis invasin protein– – Non specific DNA binding

domain from Histone H1(SPKR)4

Fortunati et al. (2000)

H fragment of the tetanus toxin DT TD GAL4 BDc GAL4 BD Box et al. (2003)scFv(FRP5) DT TD GAL4 BD GAL4 BD Uherek et al. (1998)scFv(FRP5) PE TD GAL4 BD GAL4 BD Fominaya and Wels (1996)TGF-� PE TD GAL4 BD GAL4 BD Fominaya et al. (1998)Invasin – GAL4 BD GAL4 BD Paul et al. (1997)Anti-HIV gp 120 Fab – – Protamine Chen et al. (1995)Ad5 penton protein Ad5 penton protein – Polylysine and protamine Medina-Kauwe et al. (2001a)EGF-like domain of the heregulin-�(1)

isoformAd5 penton protein – Polylysine Medina-Kauwe et al. (2001b)

Letal factor domain from anthrax toxin Letal factor domain fromanthrax toxin

GAL4 BD GAL4 BD Gaur et al. (2002)

– – T-ag NLS Human histone H1 Gaur et al. (2002)FMDV RGD sequence – �-Gald Polylysine Aris et al. (2000)FMDV RGD sequence – T-ag NLS Polylysine Aris and Villaverde (2003)– – OpT-NLS Polylysine Chan et al. (2000)Transferrin – OpT-NLS Polylysine Chan and Jans (1999)�-MSH – OpT-NLS GAL4BD Chan and Jans (2001)Galactose Fusogenic peptide

(mHA2)– Poly-L-Ornithine Nishikawa et al. (2000).

a opT-NLS, Simian Virus 40 large tumor antigen (T-Ag) derived sequence CGPGSDDEAAADAQHAAPPKKKRKVGY; T-Ag NLS, T-Ag derived sequencePKKKRKVEDPYC; �-MSH, Melanocyte stimulating hormone; DT TD, Diphteria toxin translocation domain; EGF, epidermal growth factor; A124SV40LTa,T-Ag derived sequence APPKKKRKVEDP.

b NLSV404 peptide, Simian virus SV40 large tumor antigen (T-ag) derived sequence PKKKRKVGPKKKRKVGPKKKRKVGPKKKRKVGC; GAL4BD, Saccharomyces cerevisiae GAL4 DNA binding domain; scFv(FRP5), antibody fragment specific for the tumor-associated ErbB2 antigen; DT TD, Diptheriatoxin translocation domain; PE TD, Pseudomonas exotoxin translocation domain; TGF-�, Transforming growth factor alpha; FMDV, Foot-and-mouth diseasevirus; �-MSH, Melanocyte stimulating hormone; T-Ag NLS, T-Ag derived sequence PKKKRKVEDPYC; OpT-NLS, T-Ag derived sequence CGPGSD-DEAAADAQHAAPPKKKRKVGY.

c An NLS has been described within the GAL4 BD (Silver et al., 1984; Chan et al., 1998; Chan and Jans, 1999).d A cryptic NLS has been described within the �-Gal (McInnis et al., 1995).

required to verify VP3’s selectivity in terms of tumor cellkilling.

Although not completely understood, the molecular basisof VP3’s tumor-specific nuclear localization has been inten-sively investigated (Oro and Jans, 2004; Wang et al., 2004;Poon et al., 2005a,b; Rohn et al., 2005; Wagstaff and Jans,2006). Regardless of its proapoptotic activity, understand-ing of the mechanisms and sequences responsible for VP3’stumor-cells specific nuclear localization would allow use ofthe latter as an NT module in modular transporters designedto specifically localize specifically in the nucleus of tumorbut not normal cells. The determinants of VP3 intracellu-

lar localization thus far characterized (see Figs. 3 and 4)are:

1. A bipartite basic NLS, located at the C-terminus of theprotein (aa 80-121), necessary for VP3 nuclear accu-mulation as revealed by analysis of deletion and pointmutant derivatives (Danen-van Oorschot et al., 1997,2003; Wang et al., 2004; Poon et al., 2005a,b). This motifis able to confer nuclear localization on heterologous pro-teins such as green fluorescent protein (GFP; Danen-vanOorschot et al., 2003; Wadia et al., 2004; Poon et al.,2005a,b) and maltose-binding protein (MBP; Rohn et al.,

G. Alvisi et al. / Drug Resistance Updates 9 (2006) 40–50 45

Fig. 3. Summary of the targeting sequences of VP3 and domains involved in protein–protein interaction. The single letter code is used for amino acid; NLS,nuclear localization signal; NES, nuclear export signal; PML, promyelocytic leukemia protein; Hippi, protein interactor of the Huntingtin interacting protein1; APC1, subunit of the anaphase-promoting complex/cyclosome.

2005). Intriguingly, VP3(74–121) binds to IMP�1 withthree times higher affinity compared to VP3(1–121), sug-gesting that the NLS of VP3 activity may be regulatedby intramolecular masking through the VP3 N-terminus(Wagstaff and Jans, 2006).

2. A CRM-1 recognized NES (residues 97–105) inactive intumor cells, which contributes to determine tumor cell-specificity of VP3 localization (Poon et al., 2005a). TheNES is located between the arms of the bipartite NLS,and thus aa 74–121, encompassing both the VP3 NLSand NES, represent a tNTS.

3. VP3 T108, adjacent to the NES, is specifically phos-phorylated by an unidentified kinase in tumor but notnormal cells (Rohn et al., 2002). Phosphorylation at thissite appears to be responsible for tumor cell-specificityof VP3 nuclear localization, by inhibiting NES-mediatednuclear export in tumor cells (Poon et al., 2005a), andpossibly also by enhancing its nuclear import (Rohn etal., 2002, 2005). The phosphomimetic E108 VP3 mutantderivative shows enhanced nuclear localization in normalcells, reaching levels of nuclear accumulation compara-ble to that achieved in tumor cells (Poon et al., 2005a), butis not responsive to LMB treatment, consistent with theidea that phosphorylation at T108 primarily inhibits NESactivity.

4. A leucine-rich sequence (LRS), at residues 33–46, which

nuclear matrix, associated with several functions suchas apoptosis, DNA replication and repair, transcriptionregulation and RNA transport (Takahashi et al., 2004).The LRS lies within the VP3 multimerization domain(residues 26–69; Leliveld et al., 2003a), and although pro-posed to be a putative NES (Danen-van Oorschot et al.,1997), this has been disproved (Wang et al., 2004; Poonet al., 2005a). Intriguingly, mutation of the LRS sequencenot only ablates VP3 co-localization with PML, but alsoreduces VP3 nuclear accumulation in tumor and normalcells, and LMB responsiveness in normal cells. Thus,association with PML and thereby PML NBs may be aprerequisite for VP3 nuclear export (Poon et al., 2005a).VP3 has also been reported to bind to other nuclear com-ponents such as the kinase HIPK2 (Poon et al., 2005a), thehuman death effector domain-associated factor (Danen-van Oorschot et al., 2004), DNA (Leliveld et al., 2003b)and APC-1, a subunit of the anaphase-promoting com-plex/cyclosome involved in the assembly and regulationof the cyclosome complex (Teodoro et al., 2004). VP3 hasalso been reported to interact with cytoplasmic proteinHippi, the protein interactor of the Huntingtin interactingprotein 1 (Hip-1; Cheng et al., 2003). Thus, it seems pos-sible that VP3 subcellular localization stems from several,competing processes within the cell such as nuclear andcytoplasmic retention as well as facilitated nuclear import

b

appears to mediate VP3’S interaction with the promye-locytic leukemia (PML) protein (Poon et al., 2005a), theorganizer of the PML nuclear bodies (PML NBs). PMLNBs are structured protein complexes associated with the

and export.

As mentioned above, modular transporters ideally com-ine CT, EE and NT functions in order achieve efficient

46 G. Alvisi et al. / Drug Resistance Updates 9 (2006) 40–50

Fig. 4. Model for VP3 tumor-specific nuclear localization. VP3 is recognized by IMP�1 and transported into the nucleus in both normal and tumor cells (1).Once inside the nucleus, VP3 is released from IMP�1 upon binding of RanGTP and localizes to the PML NBs (2). In tumor but not in normal cells, VP3phosphorylation on T108 prevents recognition by CRM1 (3) and export to the cytoplasm (4). Thus, VP3 localizes more strongly in the nucleus in tumor ratherthan in normal cells, as illustrated in the confocal microscopic images of 1BR3 (normal; left) and 1BR3/N (transformed; right) cells expressing GFP-VP3(lower panels); note that VP3 is, in fact, able to accumulate in the nuclei of non-tumor cells, but to a significantly reduced extent compared to in transformedcells (see Poon et al., 2005a,b).

delivery of drugs or DNA encoding toxic genes to the nucleiof target cancer cells. Importantly, VP3(74–121) not onlyexhibits tumor cell-specific nuclear localization (Poon et al.,2005a,b), but has been reported to induce apoptosis in cancercells (Danen-van Oorschot et al., 2003), as well as bind DNA(Leliveld et al., 2004), raising the possibility that this por-tion of VP3 could possess DNA binding, tumor cell-specificnuclear targeting and proapoptotic abilities. Further analysisis required to demonstrate if this is the case.

5. Tumor cell-specific phosphorylation

Studies on VP3 suggest the existence of tumor-specificcell-phosphorylation activity (Rohn et al., 2002; Zhang et al.,2004). A number of kinases show elevated activity in trans-formed cells. Protein kinase CK2 (CK2), is activated to upto 7-fold higher levels in tumor than in normal cells (Guerraand Issinger, 1999; Tawfic et al., 2001); while cytoplasmicextracts of cells obtained from SCCHN tumor surgical spec-imens show a 6-fold higher CK2 activity compared to thoseof cells obtained from normal oropharyngeal mucosa (Faustet al., 1999). These findings are interesting in light of the fact

that CK2 facilitates the nuclear accumulation of a numberof proteins, mainly through regulation of the NLS activityof a conserved modular domain known as the CcN motif(Rihs et al., 1991; Ueda et al., 1995; Longshaw et al., 2000;Briggs et al., 2001; Nuthall et al., 2002; Wilson et al., 2002;Alvisi et al., 2005). The CcN (CK2, cdc, and NLS) motifis a modular domain, originally described for T-Ag, thatcomprises phosphorylation sites for both CK2 and cyclin-dependent kinases (cdks) upstream of the NLS (Jans et al.,1991; Rihs et al., 1991). A phosphorylation site for double-stranded DNA-dependent kinase (dsDNA-PK) upstream ofthe cdk site has also been identified (Xiao et al., 1997). Invitro experiments indicate that CK2-mediated phosphoryla-tion of Ser 111/112 increases the rate of nuclear accumulationof T-Ag-CcN-containing fusion proteins (Rihs et al., 1991),by increasing the affinity of recognition by IMP�/� (Hubneret al., 1997). Phosphorylation of Thr124 by cdk34 inhibits thenuclear import process (Jans et al., 1991), while dsDNA-PKmediated phosphorylation of Ser 120 enhances the maxi-mal level of nuclear import and promotes IMP�/� bindingin synergy with the CK2 site (Xiao et al., 1997). These dataenabled to the development of a 25-amino acid optimized T-Ag NLS (opT-NLS), which has been used as an NT module

G. Alvisi et al. / Drug Resistance Updates 9 (2006) 40–50 47

in recombinant modular transporters for the specific targetingof PS agents to cancer cells to increase cell killing (Akhlyninaet al., 1997, 1999; Rosenkranz et al., 2003), or in recombi-nant modular transporter to increase reporter gene expression(Chan and Jans, 1999a,b, 2001, 2002). Since CK2 is highlyactive in tumor cells as indicated above (Faust et al., 1999),the opT-NLS itself effectively represents a tNTS, at least fortumors showing high CK2 activity. Clearly, further studiesare required to verify this interesting possibility using theapproaches described by Poon et al. (2005a,b).

6. Engineering the T-Ag CcN motif to generate atNTS

The T-Ag CcN motif has been extensively studied, withthe molecular mechanisms underlying the regulation of itsactivity well characterized (Jans et al., 1991; Rihs et al.,1991; Hubner et al., 1997; Xiao et al., 1997, 1998; Fonteset al., 2003). The introduction of point mutations substi-tuting the CK2 consensus phosphorylatin site with con-sensus sites for cAMP-dependent protein kinase (PKA) orCa2+, phospholipid-dependent protein kinase (PKC) withinthe CcN motif, results in enhanced nuclear accumulationof �-GAL, in response to agents elevating the intracellu-lar cAMP or activating PKC, respectively, when tested inaataislcspmfio

7

wtfttrtti

qt

ing molecules, including protein kinases, and even in thelevels of nuclear transport components (Kau et al., 2004;Poon and Jans, 2005). The ideal strategy for pursuing cancercell-specific targeting might therefore be to exploit as manyquantitative differences between tumor and normal cells aspossible, and use them in combination to achieve a cumulativeeffect, so that much higher concentrations of drug or suicidegene are achieved in tumor as opposed to normal cells. TheVP3 tNTS is clearly one targeting component that is muchmore efficient in conferring nuclear accumulation in tumorthan in normal cells, but this appears to be a quantitativerather than all-or-nothing effect, and hence needs to be usedin combination with other “tumor cell-enriched pathways”(e.g. phosphorylation cascades, receptor expression, endo-cytotic pathways, etc.; Guillemard and Saragovi, 2004) toachieve the equivalent of a “tumor cell-specific” effect. Thiscan also be combined with further levels of “enrichment”such as cell/tissue-specific promoters for gene therapeuticapproaches, and localized photoactivation in the case of theuse of PSs in PDT.

The VP3 tNTS and modifications thereof, as well as strate-gies inspired by the idea of tumor cell-specific phosphoryla-tion (see above), are great steps towards the long-term aimof cancer cell-specific therapies that do not kill or damagenormal cells/tissue. Clearly, however, the key will be to findother tumor cell-specific acting moieties to use in combina-teh“ncuk

8

•

•

•

•

•

n in vitro transport assay or in microinjected cells (Xiao etl., 1996, 1998). This demonstrates that the CK2 site withinhe CcN motif can be substituted by sites for other kinasesnd yet retain functionality in terms of nuclear localizationn response to specific phosphorylation; changing the kinaseite changes the cellular signal that potentially drives nuclearocalization. Substitution of the CK2 site with a potentiallyancer-specific phosphorylation site could generate a novelpecific tNTS. Screening for sequences that are selectivelyhosphorylated in the cytoplasm of tumor as opposed to nor-al cells may identify sequences that enhance tumor speci-city of modular transporters for the delivery to cancer cellsf suicide genes, or PS agents or other nuclear-acting drugs.

. Conclusions and future directions

Strategies to achieve cancer cell-specific targeting –hether at the level of the whole cell, or more specifically at

he level of nuclear transport – are obviously limited by theact that cancer cells are derived from normal cells, implyinghat much of the fundamental signaling and transport appara-us is shared between cancer and normal cells. Hence, despiteesults for VP3 and other proteins discussed here, the ideahat a targeting signal or approach could be uniquely func-ional in terms of targeting tumor as opposed to normal cellss probably naı̈ve.

As alluded above, however, it is clear that there are severaluantitative differences between cancer and normal cells inhe levels of expression and activation of different signal-

ion with these sequences. With concerted effort, the ultimatend may be in sight, not so much of a “magic bullet” per-aps, but of a multi-component entity comprising multiplesemi-magic” moieties with higher activity in tumor than inormal cells. In combination, these cancer cell hyperactiveomponents should afford a cumulative effect to achieve theltimate end of efficient and above all specific tumor cellilling.

. Summary

Modular recombinant transporters, assemblies of indepen-dently acting domains, have been shown to be capable oftargeting reporter genes or drugs to cancer cells specifi-cally through cellular targeting (CT) modules.The addition of a nuclear targeting (NT) module increasesthe ability of modular transporters to deliver suicide genesor drugs to the nucleus of the target cell.VP3 is a protein that shows tumor cell-specific nuclear tar-geting as mediated by a phosphorylation regulated tumor-specific nuclear targeting sequence (tNTS).The activity of the best characterized nuclear targeting sig-nal, the nuclear localization signal (NLS) from Simianvirus SV40 large tumor antigen (T-Ag), is increased byprotein kinase CK2 (CK2) phosphorylation upstream ofthe NLS.CK2 activity is elevated in several cancer cells so that T-Ag NLS-based modular transporters may be promising fortherapy where tumors show elevated CK2 activity.

48 G. Alvisi et al. / Drug Resistance Updates 9 (2006) 40–50

• Substitution of the CK2 site of T-Ag with consensus sitesfor kinases such Ca2+, phospholipid-dependent proteinkinase (PKC) or cAMP-dependent protein kinase (PKA)results in its activity being increased by PKC and PKAactivators, respectively. It is possible to generate tNTSs byinserting sequences phosphorylated in the cytoplasm oftumor but not normal cells in place of the T-Ag CK2 site.

• The combination of several tumor cell-specific modules,including CT and NT would increase the specificity ofdelivery to tumor cells of suicide genes or nuclear-actingdrugs.

Acknowledgments

This work was supported by the Anti-Cancer Council ofVictoria and the Australian National Health and MedicalResearch Council (fellowship #384109).

References

Akhlynina, T.V., Rosenkranz, A.A., Jans, D.A., Gulak, P.V., Serebryakova,N.V., Sobolev, A.S., 1993. The use of internalizable derivatives ofchlorin E6 for increasing its photosensitizing activity. Photochem.Photobiol. 58, 45–48.

Akhlynina, T.V., Rosenkranz, A.A., Jans, D.A., Sobolev, A.S., 1995.

A

A

A

A

A

B

B

B

B

B

Chan, C.K., Hubner, S., Hu, W., Jans, D.A., 1998. Mutual exclusivity ofDNA binding and nuclear localization signal recognition by the yeasttranscription factor GAL4: implications for nonviral DNA delivery.Gene Ther. 5, 1204–1212.

Chan, C.K., Jans, D.A., 1999a. Enhancement of polylysine-mediatedtransferrinfection by nuclear localization sequences: polylysine doesnot function as a nuclear localization sequence. Hum. Gene Ther. 10,1695–1702.

Chan, C.K., Jans, D.A., 1999b. Synergy of importin alpha recognitionand DNA binding by the yeast transcriptional activator GAL4. FEBSLett. 462, 221–224.

Chan, C.K., Senden, T., Jans, D.A., 2000. Supramolecular structure andnuclear targeting efficiency determine the enhancement of transfectionby modified polylysines. Gene Ther. 7, 1690–1697.

Chan, C.K., Jans, D.A., 2001. Enhancement of MSH receptor- and GAL4-mediated gene transfer by switching the nuclear import pathway. GeneTher. 8, 166–171.

Chan, C.K., Jans, D.A., 2002. Using nuclear targeting signals to enhancenon-viral gene transfer. Immunol. Cell Biol. 80, 119–130.

Chen, S.Y., Zani, C., Khouri, Y., Marasco, W.A., 1995. Design of agenetic immunotoxin to eliminate toxin immunogenicity. Gene Ther.2, 116–123.

Cheng, C.M., Huang, S.P., Chang, Y.F., Chung, W.Y., Yuo, C.Y., 2003.The viral death protein Apoptin interacts with Hippi, the proteininteractor of Huntingtin-interacting protein 1. Biochem. Biophys. Res.Commun. 305, 359–364.

Danen-van Oorschot, A.A., Fischer, D.F., Grimbergen, J.M., Klein, B.,Zhuang, S., Falkenburg, J.H., Backendorf, C., Quax, P.H., Van derEb, A.J., Noteborn, M.H., 1997. Apoptin induces apoptosis in humantransformed and malignant cells but not in normal cells. Proc. Natl.Acad. Sci. U.S.A. 94, 5843–5847.

D

D

D

E

F

F

F

F

F

F

Insulin-mediated intracellular targeting enhances the photodynamicactivity of chlorin e6. Cancer Res. 55, 1014–1019.

khlynina, 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. Nucleartargeting of chlorin e6 enhances its photosensitizing activity. J. Biol.Chem. 272, 20328–20331.

khlynina, 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 enhancenuclear delivery and photodynamic action of internalizable conjugatescontaining chlorin e6. Int. J. Cancer 81, 734–740.

lvisi, G., Jans, D.A., Guo, J., Pinna, L.A., Ripalti, A., 2005. A proteinkinase CK2 site flanking the nuclear targeting signal enhances nucleartransport of human cytomegalovirus ppUL44. Traffic 6, 1002–1013.

ris, A., Feliu, J.X., Knight, A., Coutelle, C., Villaverde, A., 2000.Exploiting viral cell-targeting abilities in a single polypeptide, non-infectious, recombinant vehicle for integrin-mediated DNA deliveryand gene expression. Biotechnol. Bioeng. 68, 689–696.

ris, A., Villaverde, A., 2003. Engineering nuclear localization signals inmodular protein vehicles for gene therapy. Biochem. Biophys. Res.Commun. 304, 625–631.

island, S.K., Singh, D., Gariepy, J., 1999. Potentiation of chlorin e6 pho-todynamic activity in vitro with peptide-based intracellular vehicles.Bioconjug. Chem. 10, 982–992.

ox, M., Parks, D.A., Knight, A., Hale, C., Fishman, P.S., Fairweather,N.F., 2003. A multi-domain protein system based on the HC fragmentof tetanus toxin for targeting DNA to neuronal cells. J. Drug Target11, 333–343.

ranton, P.E., Roopchand, D.E., 2001. The role of adenovirus E4orf4protein in viral replication and cell killing. Oncogene 20, 7855–7865.

riggs, L.J., Johnstone, R.W., Elliot, R.M., Xiao, C.Y., Dawson, M., Tra-pani, J.A., Jans, D.A., 2001. Novel properties of the protein kinaseCK2-site-regulated nuclear- localization sequence of the interferon-induced nuclear factor IFI 16. Biochem. J. 353, 69–77.

rokx, R.D., Bisland, S.K., Gariepy, J., 2002. Designing peptide-basedscaffolds as drug delivery vehicles. J. Control. Release 78, 115–123.

anen-van Oorschot, A.A., Zhang, Y.H., Leliveld, S.R., Rohn, J.L., See-len, M.C., Bolk, M.W., Van Zon, A., Erkeland, S.J., Abrahams, J.P.,Mumberg, D., Noteborn, M.H., 2003. Importance of nuclear local-ization of apoptin for tumor-specific induction of apoptosis. J. Biol.Chem. 278, 27729–27736.

anen-van Oorschot, A.A., Voskamp, P., Seelen, M.C., van Miltenburg,M.H., Bolk, M.W., Tait, S.W., Boesen-de Cock, J.G., Rohn, J.L.,Borst, J., Noteborn, M.H., 2004. Human death effector domain-associated factor interacts with the viral apoptosis agonist Apoptinand exerts tumor-preferential cell killing. Cell Death Differ. 11, 564–573.

e Clercq, E., 2004. Antivirals and antiviral strategies. Nat. Rev. Micro-biol. 2, 704–720.

verts, B., van der Poel, H.G., 2005. Replication-selective oncolyticviruses in the treatment of cancer. Cancer Gene Ther. 12, 141–161.

aust, R.A., Niehans, G., Gapany, M., Hoistad, D., Knapp, D., Cherwitz,D., Davis, A., Adams, G.L., Ahmed, K., 1999. Subcellular immunolo-calization of protein kinase CK2 in normal and carcinoma cells. Int.J. Biochem. Cell. Biol. 31, 941–949.

ominaya, J., Uherek, C., Wels, W., 1998. A chimeric fusion proteincontaining transforming growth factor-alpha mediates gene transfervia binding to the EGF receptor. Gene Ther. 5, 521–530.

ominaya, J., Wels, W., 1996. Target cell-specific DNA transfer mediatedby a chimeric multidomain protein. Novel non-viral gene deliverysystem. J. Biol. Chem. 271, 10560–10568.

ontes, M.R., Teh, T., Toth, G., John, A., Pavo, I., Jans, D.A., Kobe,B., 2003. Role of flanking sequences and phosphorylation in therecognition of the simian-virus-40 large T-antigen nuclear localiza-tion sequences by importin-alpha. Biochem. J. 375, 339–349.

ortunati, E., Ehlert, E., van Loo, N.D., Wyman, C., Eble, J.A., Grosveld,F., Scholte, B.J., 2000. A multi-domain protein for beta1 integrin-targeted DNA delivery. Gene Ther. 7, 1505–1515.

ukuda, M., Asano, S., Nakamura, T., Adachi, M., Yoshida, M., Yanagida,M., Nishida, E., 1997. CRM1 is responsible for intracellular transportmediated by the nuclear export signal. Nature 390, 308–311.

G. Alvisi et al. / Drug Resistance Updates 9 (2006) 40–50 49

Gaur, R., Gupta, P.K., Goyal, A., Wels, W., Singh, Y., 2002. Deliveryof nucleic acid into mammalian cells by anthrax toxin. Biochem.Biophys. Res. Commun. 297, 1121–1127.

Gijsens, A., Missiaen, L., Merlevede, W., de Witte, P., 2000. Epidermalgrowth factor-mediated targeting of chlorin e6 selectively potentiatesits photodynamic activity. Cancer Res. 60, 2197–2202.

Glover, D.J., Lipps, H.J., Jans, D.A., 2005. Towards safe, non-viral ther-apeutic gene expression in humans. Nat. Rev. Genet. 6, 299–310.

Guelen, L., Paterson, H., Gaken, J., Meyers, M., Farzaneh, F., Tavassoli,M., 2004. TAT-apoptin is efficiently delivered and induces apoptosisin cancer cells. Oncogene 23, 1153–1165.

Guerra, B., Issinger, O.G., 1999. Protein kinase CK2 and its role incellular proliferation, development and pathology. Electrophoresis 20,391–408.

Guillemard, V., Saragovi, H.U., 2004. Novel approaches for targeted can-cer therapy. Curr. Cancer Drug Targets 4, 313–326.

Hacein-Bey-Abina, S., von Kalle, C., Schmidt, M., Le Deist, F., Wulffraat,N., McIntyre, E., Radford, I., Villeval, J.L., Fraser, C.C., Cavazzana-Calvo, M., Fischer, A., 2003a. A serious adverse event after successfulgene therapy for X-linked severe combined immunodeficiency. N.Engl. J. Med. 348, 255–256.

Hacein-Bey-Abina, S., Von Kalle, C., Schmidt, M., McCormack, M.P.,Wulffraat, N., Leboulch, P., et al., 2003b. LMO2-associated clonalT cell proliferation in two patients after gene therapy for SCID-X1.Science 302, 415–419.

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.

Jans, D.A., Ackermann, M.J., Bischoff, J.R., Beach, D.H., Peters, R.,1991. p34cdc2-mediated phosphorylation at T124 inhibits nuclear

J

K

K

K

L

L

L

L

L

L

Lutsenko, S.V., Feldman, N.B., Finakova, G.V., Posypanova, G.A., Sev-erin, S.E., Skryabin, K.G., Kirpichnikov, M.P., Lukyanets, E.A.,Vorozhtsov, G.N., 1999. Targeting phthalocyanines to tumor cellsusing epidermal growth factor conjugates. Tumour Biol. 20, 218–224.

MacFarlane, M., 2003. TRAIL-induced signalling and apoptosis. Toxicol.Lett. 139, 89–97.

Mathis, J.M., Stoff-Khalili, M.A., Curiel, D.T., 2005. Oncolyticadenoviruses-selective retargeting to tumor cells. Oncogene 24,7775–7791.

McInnis, R., Perkinson, R.A., Kuo, B.A., Norton, P.A., 1995. Unexpectednuclear localization of a chimeric beta-galactosidase lacZ reportergene product in mammalian cells. Biochem. Mol. Biol. Int. 36,483–490.

Medina-Kauwe, L.K., Kasahara, N., Kedes, L., 2001a. 3PO, a novel non-viral gene delivery system using engineered Ad5 penton proteins.Gene Ther. 8, 795–803.

Medina-Kauwe, L.K., Maguire, M., Kasahara, N., Kedes, L., 2001b. Non-viral gene delivery to human breast cancer cells by targeted Ad5penton proteins. Gene Ther. 8, 1753–1761.

Meyer, B.E., Meinkoth, J.L., Malim, M.H., 1996. Nuclear transport ofhuman immunodeficiency virus type 1, visna virus, and equine infec-tious anemia virus Rev proteins: identification of a family of trans-ferable nuclear export signals. J. Virol. 70, 2350–2359.

Miron, M.J., Gallouzi, I.E., Lavoie, J.N., Branton, P.E., 2004. Nuclearlocalization of the adenovirus E4orf4 protein is mediated throughan arginine-rich motif and correlates with cell death. Oncogene 23,7458–7468.

Nishikawa, M., Yamauchi, M., Morimoto, K., Ishida, E., Takakura, Y.,Hashida, M., 2000. Hepatocyte-targeted in vivo gene expression byintravenous injection of plasmid DNA complexed with synthetic multi-functional gene delivery system. Gene Ther. 7, 548–555.

N

N

O

P

P

P

P

P

P

R

R

import of SV-40 T antigen proteins. J. Cell Biol. 115, 1203–1212.

ori, G., 1996. Tumour photosensitizers: approaches to enhance the selec-tivity and efficiency of photodynamic therapy. J. Photochem. Photo-biol. B. 36, 87–93.

alderon, D., Richardson, W.D., Markham, A.F., Smith, A.E., 1984.Sequence requirements for nuclear location of simian virus 40 large-Tantigen. Nature 311, 33–38.

au, T.R., Way, J.C., Silver, P.A., 2004. Nuclear transport and cancer:from mechanism to intervention. Nat. Rev. Cancer 4, 106–117.

udo, N., Wolff, B., Sekimoto, T., Schreiner, E.P., Yoneda, Y., Yanagida,M., Horinouchi, S., Yoshida, M., 1998. Leptomycin B inhibition ofsignal-mediated nuclear export by direct binding to CRM1. Exp. CellRes. 242, 540–547.

avoie, 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.

eliveld, S.R., Zhang, Y.H., Rohn, J.L., Noteborn, M.H., Abrahams, J.P.,2003a. Apoptin induces tumor-specific apoptosis as a globular multi-mer. J. Biol. Chem. 278, 9042–9051.

eliveld, S.R., Dame, R.T., Mommaas, M.A., Koerten, H.K., Wyman,C., Danen-van Oorschot, A.A., Rohn, J.L., Noteborn, M.H., Abra-hams, J.P., 2003b. Apoptin protein multimers form distinct higher-order nucleoprotein complexes with DNA. Nucleic Acids Res. 31,4805–4813.

eliveld, S.R., Dame, R.T., Rohn, J.L., Noteborn, M.H., Abrahams,J.P., 2004. Apoptin’s functional N- and C-termini independently bindDNA. FEBS Lett. 557, 155–158.

iu, Q., Hamblin, M.R., 2005. Macrophage-targeted photodynamic ther-apy: scavenger receptor expression and activation state. Int. J.Immunopathol. Pharmacol. 18, 391–402.

ongshaw, V.M., Dirr, H.W., Blatch, G.L., Lassle, M., 2000. The in vitrophosphorylation of the co-chaperone mSTI1 by cell cycle kinasessubstantiates a predicted casein kinase II-p34cdc2-NLS (CcN) motif.Biol. Chem. 381, 1133–1138.

oteborn, M.H., Todd, D., Verschueren, C.A., de Gauw, H.W., Curran,W.L., Veldkamp, S., Douglas, A.J., McNulty, M.S., van der Eb, A.,Koch, G., 1994. A single chicken anemia virus protein induces apop-tosis. J. Virol. 68, 346–351.

uthall, H.N., Joachim, K., Palaparti, A., Stifani, S., 2002. A role for cellcycle-regulated phosphorylation in Groucho-mediated transcriptionalrepression. J. Biol. Chem. 277, 51049–51057.

ro, C., Jans, D.A., 2004. The tumour specific pro-apoptotic factorapoptin (Vp3) from chicken anaemia virus. Curr. Drug Targets 5,179–190.

aul, R.W., Weisser, K.E., Loomis, A., Sloane, D.L., LaFoe, D., Atkinson,E.M., Overell, R.W., 1997. Gene transfer using a novel fusion protein,GAL4/invasin. Hum. Gene Ther. 8, 1253–1262.

eng, Q., Moan, J., Nesland, J.M., 1996. Correlation of subcellular andintratumoral photosensitizer localization with ultrastructural featuresafter photodynamic therapy. Ultrastruct. Pathol. 20, 109–129.

ilger, B.D., Perozzo, R., Alber, F., Wurth, C., Folkers, G., Scapozza,L., 1999. Substrate diversity of herpes simplex virus thymidinekinase. Impact of the kinematics of the enzyme. J. Biol. Chem. 274,31967–31973.

oon, I.K., Jans, D.A., 2005. Regulation of nuclear transport: central rolein development and transformation? Traffic 6, 173–186.

oon, I.K., Oro, C., Dias, M.M., Zhang, J., Jans, D.A., 2005a. Apoptinnuclear accumulation is modulated by a CRM1-recognized nuclearexport signal that is active in normal but not in tumor cells. CancerRes. 65, 7059–7064.

oon, I.K., Oro, C., Dias, M.M., Zhang, J.P., Jans, D.A., 2005b. Atumor cell-specific nuclear targeting signal within chicken anemiavirus VP3/apoptin. J. Virol. 79, 1339–1341.

ainov, N.G., Ren, H., 2003. Clinical trials with retrovirus mediated genetherapy—what have we learned? J. Neurooncol. 65, 227–236.

aper, S.E., Chirmule, N., Lee, F.S., Wivel, N.A., Bagg, A., Gao, G.P.,Wilson, J.M., Batshaw, M.L., 2003. Fatal systemic inflammatoryresponse syndrome in a ornithine transcarbamylase deficient patientfollowing adenoviral gene transfer. Mol. Genet. Metab. 80, 148–158.

50 G. Alvisi et al. / Drug Resistance Updates 9 (2006) 40–50

Rihs, H.P., Jans, D.A., Fan, H., Peters, R., 1991. The rate of nuclearcytoplasmic protein transport is determined by the casein kinase IIsite flanking the nuclear localization sequence of the SV40 T-antigen.EMBO J. 10, 633–639.

Ritter, W., Plank, C., Lausier, J., Rudolph, C., Zink, D., Reinhardt,D., Rosenecker, J., 2003. A novel transfecting peptide comprisinga tetrameric nuclear localization sequence. J. Mol. Med. 81, 708–717.

Rohn, J.L., Zhang, Y.H., Aalbers, R.I., Otto, N., Den Hertog, J., Hen-riquez, N.V., Van De Velde, C.J., Kuppen, P.J., Mumberg, D., Donner,P., Noteborn, M.H., 2002. A tumor-specific kinase activity regulatesthe viral death protein Apoptin. J. Biol. Chem. 277, 50820–50827.

Rohn, J.L., Noteborn, M.H., 2004. The viral death effector Apoptinreveals tumor-specific processes. Apoptosis 9, 315–322.

Rohn, J.L., Zhang, Y.H., Leliveld, S.R., Danen-van Oorschot, A.A., Hen-riquez, N.V., Abrahams, J.P., Noteborn, M.H., 2005. Relevance ofapoptin’s integrity for its functional behavior. J. Virol. 79, 1337–1338.

Rosenkranz, A.A., Lunin, V.G., Gulak, P.V., Sergienko, O.V., Shumiant-seva, 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 modulartransporters for cell-specific nuclear delivery of locally acting drugsenhance photosensitizer activity. FASEB J. 17, 1121–1123.

Seth, P., 1994. Mechanism of adenovirus-mediated endosome lysis: roleof the intact adenovirus capsid structure. Biochem. Biophys. Res.Commun. 205, 1318–1324.

Sharman, W.M., Allen, C.M., van Lier, J.E., 1999. Photodynamic thera-peutics: basic principles and clinical applications. Drug Discov. Today4, 507–517.

Silver, P.A., Keegan, L.P., Ptashine, M., 1984. Amino terminus of theyeast GAL4 gene product is sufficient for nuclear localization. Proc.Natl. Acad. Sci. U.S.A. 81, 5951–5955.

Singh, D., Bisland, S.K., Kawamura, K., Gariepy, J., 1999. Peptide-based

S

T

T

T

T

U

U

V

Wadia, J.S., Wagner, M.V., Ezhevsky, S.A., Dowdy, S.F., 2004.Apoptin/VP3 contains a concentration-dependent nuclear localiza-tion signal (NLS), not a tumorigenic selective NLS. J. Virol. 78,6077–6078.

Wagstaff, K.M., Jans, D.A., 2006. Intramolecular masking of nuclearlocalization signals: analysis of importin binding using a novelAlphaScreen-based method. Anal. Biochem. 348, 49–56.

Walczak, H., Miller, R.E., Ariail, K., Gliniak, B., Griffith, T.S., Kubin,M., et al., 1999. Tumoricidal activity of tumor necrosis factor-relatedapoptosis-inducing 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 putativeNES mediates cytoplasmic localization of Apoptin in normal cells.Acta Biochim. Biophys. Sin. (Shanghai) 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, fea-turing the uses of surveillance data for cancer prevention and control.J. Natl. Cancer Inst. 95, 1276–1299.

Weis, K., 1998. Importins and exportins: how to get in and out of thenucleus. Trends Biochem. Sci. 23, 185–189.

Wiedmann, M., Caca, K., Berr, F., Schiefke, I., Tannapfel, A., Wit-tekind, C., Mossner, J., Hauss, J., Witzigmann, H., 2003. Neoadjuvantphotodynamic therapy as a new approach to treating hilar cholangio-carcinoma: a phase II pilot study. Cancer 97, 2783–2790.

Williams, D.A., Baum, C., 2003. Medicine. Gene therapy—new chal-lenges ahead. Science 302, 400–401.

Wilson, K.C., Cruikshank, W.W., Center, D.M., Zhang, Y., 2002.Prointerleukin-16 contains a functional CcN motif that regulatesnuclear localization. Biochemistry 41, 14306–14312.

Xiao, C.Y., Hubner, S., Elliot, R.M., Caon, A., Jans, D.A., 1996. A con-sensus cAMP-dependent protein kinase (PK-A) site in place of the

X

X

Y

Z

Z

intracellular shuttle able to facilitate gene transfer in mammalian cells.Bioconjug. Chem. 10, 745–754.

obolev, A.S., Jans, D.A., Rosenkranz, A.A., 2000. Targeted intracellulardelivery of photosensitizers. Prog. Biophys. Mol. Biol. 73, 51–90.

akahashi, Y., Lallemand-Breitenbach, V., Zhu, J., de The, H., 2004. PMLnuclear bodies and apoptosis. Oncogene 23, 2819–2824.

avassoli, M., Guelen, L., Luxon, B.A., Gaken, J., 2005. Apoptin: specifickiller of tumor cells? Apoptosis 10, 717–724.

awfic, S., Yu, S., Wang, H., Faust, R., Davis, A., Ahmed, K., 2001. Pro-tein kinase CK2 signal in neoplasia. Histol. Histopathol. 16, 573–582.

eodoro, J.G., Heilman, D.W., Parker, A.E., Green, M.R., 2004. The viralprotein Apoptin associates with the anaphase-promoting complex toinduce G2/M arrest and apoptosis in the absence of p53. Genes Dev.18, 1952–1957.

eda, H., Ullrich, S.J., Gangemi, J.D., Kappel, C.A., Ngo, L., Feitelson,M.A., Jay, G., 1995. Functional inactivation but not structural mutationof p53 causes liver cancer. Nat. Genet. 9, 41–47.

herek, C., Fominaya, J., Wels, W., 1998. A modular DNA carrier proteinbased on the structure of diphtheria toxin mediates target cell-specificgene delivery. J. Biol. Chem. 273, 8835–8841.

assaux, G., Martin-Duque, P., 2004. Use of suicide genes for cancergene therapy: study of the different approaches. Expert Opin. Biol.Ther. 4, 519–530.

CcN motif casein kinase II site simian virus 40 large T-antigen con-fers PK-A-mediated regulation of nuclear import. J. Biol. Chem. 271,6451–6457.

iao, C.Y., Hubner, S., Jans, D.A., 1997. SV40 large tumor antigennuclear import is regulated by the double-stranded DNA-dependentprotein kinase site (serine 120) flanking the nuclear localizationsequence. J. Biol. Chem. 272, 22191–22198.

iao, C.Y., Jans, P., Jans, D.A., 1998. Negative charge at the proteinkinase CK2 site enhances recognition of the SV40 large T-antigenNLS by importin: effect of conformation. FEBS Lett. 440, 297–301.

oung, L.S., Searle, P.F., Onion, D., Mautner, V., 2006. Viral gene therapystrategies: from basic science to clinical application. J. Pathol. 208,299–318.

hang, Y.H., Kooistra, K., Pietersen, A., Rohn, J.L., Noteborn, M.H.,2004. Activation of the tumor-specific death effector apoptin and itskinase by an N-terminal determinant of simian virus 40 large T anti-gen. J. Virol. 78, 9965–9976.

huang, S.M., Landegent, J.E., Verschueren, C.A., Falkenburg, J.H., vanOrmondt, H., van der Eb, A.J., Noteborn, M.H., 1995. Apoptin, aprotein encoded by chicken anemia virus, induces cell death in varioushuman hematologic malignant cells in vitro. Leukemia 9 (Suppl. 1),S118–S120.