apoptin nuclear accumulation is modulated by a crm1 ... · apoptin nuclear accumulation is...

Apoptin Nuclear Accumulation Is Modulated by a CRM1-Recognized

Nuclear Export Signal that Is Active in Normal

but not in Tumor Cells

Ivan K.H. Poon,1Cristina Oro,

1Manisha M. Dias,

1Jingpu Zhang,

3and David A. Jans

1,2

1Nuclear Signaling Laboratory, Department of Biochemistry and Molecular Biology, Monash University; 2ARC Centre of Excellence forBiotechnology and Development, Clayton, Victoria, Australia; and 3Center for Developmental Biology, Institute of Genetics andDevelopmental Biology, Chinese Academy of Sciences, Beijing, P.R. China

Abstract

Tumor cell–specific activity of chicken anemia virus viralprotein 3 (VP3 or apoptin) is believed to be dependent on itsability to localize in the nucleus of transformed but not ofprimary or nontransformed cells. The present study charac-terizes the signals responsible for the novel nucleocytoplasmictrafficking properties of VP3 using two isogenic tumor/nontumor cell pairs. In addition to the tumor cell–specificnuclear targeting signal, comprising two stretches of basicamino acids in the VP3 COOH terminus which are highlyefficient in tumor but not in normal cells, we define the CRM1-recognized nuclear export sequence (NES) within the VP3tumor cell–specific nuclear targeting signal for the first time.Intriguingly, the NES (amino acids 97-105) is functional innormal but not in tumor cells through the action of thethreonine 108 phosphorylation site adjacent to the NES whichinhibits its action. In addition, we characterize a leucine-richsequence (amino acids 33-46) that assists VP3 nuclearaccumulation by functioning as a nuclear retention sequence,conferring association with promyelocytic leukemia nuclearbodies. This unique combination of signals is the basis of thetumor cell–specific nuclear targeting abilities of VP3. (CancerRes 2005; 65(16): 7059-64)

Introduction

Chicken anemia virus viral protein 3 (VP3 or apoptin) has beenreported to possess tumor cell–specific proapoptotic activity,integrally linked to its ability to localize in the nucleus of trans-formed cells but not of primary or nontransformed cells (1–4).Conclusions about VP3 tumor cell–specific nuclear targeting andproapoptotic activities, however, have thus far been based largelyon analysis of nonisogenic cells (1). VP3 properties in SAOS-2human osteosarcoma cells, for example, have often been comparedwith those of VH10 normal human skin fibroblasts (2–4), making itessentially impossible to conclude that the differential properties ofVP3 in tumor/transformed cells are attributable to tumorigenicstatus, rather than to any number of other differences between thecell types used (1).The tumor cell–specific nuclear targeting ability of VP3 can

only be rigorously established through quantitative analyses of

isogenic cell pairs at the single cell level. As a first step towardsthis goal, we recently analyzed the nuclear targeting abilities ofVP3 in two different isogenic cell pairs, and showed that VP3indeed harbors a tumor cell–specific nuclear targeting signal inits COOH terminus (5). Through the use of truncation and pointmutants of VP3, the present study builds on this work, defining,for the first time, the VP3 tumor cell–specific nuclear targetingsignal within residues 74 to 121, which contain the basic ‘‘NLS1’’and ‘‘NLS2’’ nuclear localization sequences (NLS; see Fig. 1A). Italso defines the roles of the leucine-rich sequence (LRS) at aminoacids 33 to 46, the nuclear export signal (NES) at amino acids97 to 105, and the threonine 108 (T108) phosphorylation site,which is known to be phosphorylated specifically in tumor butnot in normal cells (6). The results show for the first time thatthe tumor cell–specific nuclear localization ability of VP3 is theproduct of a set of novel targeting sequences; the key elementis the CRM1-recognized VP3 NES which, through the inhibitoryaction of T108, operates uniquely in normal but not in tumorcells.

Materials and Methods

Expression constructs. Plasmid pEPI (7) was provided by H.J. Lipps andplasmids pEGFP-PML-C3 and pEGFP-HIPK2-C3 by A. Thorburn (8).

Chicken anemia virus VP3 encoding plasmid constructs were prepared in

the Gateway system (5), with VP3-encoding fragments generated by PCR

using platinum Taq DNA polymerase (Invitrogen, Mt. Waverly, Victoria,Australia). Site-directed mutagenesis was done using the QuikChange site-

directed mutagenesis system (Stratagene, La Jolla, CA). The fidelity of

plasmid constructs was confirmed by DNA sequencing.

Mammalian cell culture and transfection. SAOS-2 and SR40 cells werecultured in McCoy’s media (JRH Bioscience, Brooklyn, Victoria, Australia),

and COS-7 and CV-1 cells in DMEM, supplemented with 10% FCS in a

humidified incubator (Heraeus Instruments, Hanau, Hessen, Germany) in a5% CO2 atmosphere at 37jC. Cells were transfected 24 hours after plating

on coverslips using TransIT Transfection Reagent (Mirus, Madison, WI)

according to the specifications of the manufacturer.

Immunofluorescence. Immunostaining for promyelocytic leukemia(PML) was carried out on 4% paraformaldehyde–fixed, 0.25% Triton X-

100–permeabilized, bovine serum albumin–blocked cells (9, 10) using

specific anti-PML primary (Santa Cruz, Los Angeles, CA) and Alexa 546–

coupled secondary (Invitrogen) antibodies.Confocal laser scanning microscopy and image analysis. Cells were

imaged by confocal laser scanning microscopy (CLSM) using Bio-Rad

MRC-600 CLSM (5, 9, 10) or Perkin-Elmer Ultra-view. For the former, a40� water immersion objective was used for live-cell imaging on a heated

stage. In the case of the Ultra-view, a 100� oil immersion objective was

used for both live (heated stage) and fixed cell imaging. CLSM images

were analyzed using the Image J 1.62 public domain software. The ratio(Fn/c) of nuclear fluorescence (Fn) to cytoplasmic fluorescence (Fc) was

determined, subsequent to the subtraction of background fluorescence

(5, 9, 10).

Requests for reprints: David A. Jans, Nuclear Signaling Laboratory, Department ofBiochemistry and Molecular Biology, Monash University, P.O. Box 13D, Clayton,Victoria 3800, Australia. Phone: 00613/99053778; Fax: 00613/99054699; E-mail: [email protected].

I2005 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-05-1370

www.aacrjournals.org 7059 Cancer Res 2005; 65: (16). August 15, 2005

Priority Report

Research. on October 11, 2020. © 2005 American Association for Cancercancerres.aacrjournals.org Downloaded from

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Results

VP3 localizes in the nucleusmore efficiently in tumor than innormal isogenic cells. Two isogenic cell pairs identical in genotype,except for their transformed/nontransformed status, were selectedto examine VP3 subcellular localization: the tumorigenic SAOS-2line mutated in the retinoblastoma tumor suppressor gene product,together with its nontransformed SR40 counterpart derived bytransfection of SAOS-2 with the full-length retinoblastoma cDNA(11, 12), and CV-1 African green monkey kidney cells (non-transformed) together with the SV40 virus–transformed derivativeCOS-7 line (13). Cells were transfected to express green fluorescent

protein (GFP) or the various GFP-VP3 fusion constructs, and imaged

live 16 hours later using CLSM. Full-length VP3 was found to confer

nuclear localization on GFP in the transformed SAOS-2 and COS-7

lines (Fig. 1B and C), but in contrast to previous reports (2–4; see,

however, refs. 5, 14), it also localized in the nucleus in both

nontransformed cell types (SR40 and CV-1). Determination of the

nuclear to cytoplasmic ratio (Fn/c) by image analysis revealed that

the nontransformed lines accumulated GFP-VP3(1-121) to signifi-

cantly (2-fold) lower levels than their transformed counterparts

(P < 0.002; Fig. 1B and C). VP3 thus localizes to a greater extent in

the nucleus of transformed compared with nontransformed cells.

Figure 1. Nuclear accumulation of VP3 isdependent on the COOH-terminal NLS1and NLS2 sequences. A, schematicdiagram of targeting sequences within VP3(single-letter amino acid code), highlightingthe putative NLSs (NLS1, amino acids82-88; NLS2, amino acids 111-121), thetwo potential leucine-rich NESs (LRS,amino acids 33-46; NES, amino acids97-105), and the phosphorylation site(T108). B, CLSM images of SAOS-2 cells(top rows ) and SR40 cells (bottom rows ),16 hours posttreatment to express theindicated GFP-VP3 fusion proteins, andquantitative analysis (bottom ) of the levelsof nuclear accumulation [Fn/c, ratio of thenuclear fluorescence (Fn) to thecytoplasmic fluorescence (Fc) after thesubtraction of background fluorescence],as determined using the Image J publicdomain image analysis software aspreviously described (10), from CLSMimages such as those shown. Columns,mean Fn/c (n z 24); bars, SE. Significantdifferences (P values) for the Fn/c valuesbetween SAOS-2 and SR40 cells areindicated. C, CLSM images of COS-7 cells(leftmost ) and CV-1 cells (right ) 16 hoursposttreatment to express the indicatedGFP-VP3 fusions and quantitative analysisof the levels of nuclear accumulation fromCLSM images such as those shown(right ). Columns, mean Fn/c (n z 15);bars, SE. Significant differences(P values) for the Fn/c values betweenCOS-7 and CV-1 cells are indicated.

Cancer Research

Cancer Res 2005; 65: (16). August 15, 2005 7060 www.aacrjournals.org



Definition of the VP3 tumor cell–specific nuclear targetingsignal. To define the responsible sequences, GFP-VP3 truncatedderivatives were initially examined. COOH-terminal truncations ofeven the last 10 amino acids of VP3, deleting NLS2 specifically,were found to confer predominantly cytoplasmic localization onGFP in the four cell lines (Fig. 1, and data not shown), resulting inan Fn/c < 1 (Fig. 1B and C). This was in contrast to the strongnuclear accumulation (Fn/c of f20) in SAOS-2 and COS-7 cells ofGFP-VP3(74-121) containing both NLS1 and NLS2: significantlyhigher (P < 0.0005) than in their nontransformed isogeniccounterparts (Fn/c values of f11 and 4 in SR40 and CV-1 cells,respectively). GFP-VP3(104-121), containing only NLS2, showeddiffuse nuclear and cytoplasmic localization similar to that ofGFP alone (Fig. 1C), indicating that NLS2 alone was not sufficientfor nuclear accumulation. Finally, point mutations in either NLS(KK86/87 to NN in NLS1m, single-letter amino acid code, andRR117/118 to NN in NLS2m) abrogated nuclear accumulation in thecontext of full-length VP3 (Fig. 1B), demonstrating that bothNLS1 and NLS2 are required for tumor cell–specific nucleartargeting. VP3 NLS1 and NLS2 thus constitute the tumor cell–specific nuclear targeting signal that is highly efficient in nucleartargeting in transformed but not in nontransformed cells. Thatcells expressing the NLS1- and NLS2-mutated GFP-VP3 deriva-tives, in contrast to the other proteins analyzed, showed discretefoci in the cytoplasm (see Fig. 1B) most likely relates to thereported ability of VP3 to aggregate/multimerize in the cytoplasmwhen not imported into the nucleus (e.g., in nontumor cells;ref. 4).VP3 leucine-rich sequences differentially modulate VP3

nuclear targeting. To assess the contribution of the leucine-richLRS and NES sequences (see Fig. 1A) to VP3 subcellular localization,localization of the GFP-VP3 constructs was examined in theabsence and presence of a specific inhibitor of the nuclearexport receptor CRM1, leptomycin B (Fig. 2B ; ref. 15). In a similarfashion to its effect on the GFP-Rev control molecule containingthe CRM1-recognized HIV-1 Rev protein NES, leptomycin Btreatment significantly (P < 0.0001) increased nuclear accumulationof GFP-VP3(1-121) in SR40 cells, the Fn/c value of f50 in itspresence being equivalent to that in SAOS-2 cells in the absence ofleptomycin B (Fig. 2B). In contrast, GFP-VP3(1-121) nuclearaccumulation was not increased by leptomycin B in SAOS-2 cells,implying that CRM1-dependent nuclear export occurs differentiallyin the two isogenic lines; comparable differential effects ofleptomycin B were also observed in COS-7/CV-1 cells (data notshown).Point mutations within the two putative leucine-rich NESs had

diverse effects; substitution of LI104/5 to AG (NESm) significantlyincreased (P < 0.0001) nuclear accumulation in SR40 but not inSAOS-2 cells, whereas mutations within the LRS at amino acids33 to 46 (LRSm) resulted in significantly (P < 0.0001) reducednuclear accumulation in both lines. The implication was that theCOOH-terminal NES is functional and responsible in part for thereduced nuclear accumulation of VP3 in SR40 as opposed toSAOS-2 cells, whereas the NH2-terminal LRS is not a functionalNES, but rather enhances nuclear import, possibly throughfacilitating nuclear retention (see below). That the LRS contrib-utes strongly to VP3 nuclear localization is consistent with thefact that GFP-VP3(74-121), lacking the LRS, shows nuclearlocalization levels only about half of those of GFP-VP3(1-121)(see Fig. 1B and C). VP3 thus possesses a Crm1-recognized NESthat is active in normal but not in tumor cells, in addition to an

LRS that facilitates nuclear accumulation in normal and tumorcells.Threonine 108 inhibits nuclear export of VP3 in normal but

not in tumor cells. The possibility that phosphorylation of T108

in SAOS-2 cells (6) could regulate VP3 NES activity wasaddressed by examining the nuclear targeting properties andleptomycin B sensitivity of A108- and E108-substituted derivatives ofGFP-VP3(1-121). The E108 derivative, with the negatively chargedresidue approximating T108-prephosphorylated VP3, was found toaccumulate in the nucleus of SR40 cells to a level comparable tothat of the wild-type protein in SAOS-2 cells (and to that of theNES mutant in both cell lines). No increase in nuclearlocalization of the derivative was effected by leptomycin B(Fig. 2B), consistent with the idea that phosphorylation of VP3at T108 inhibits NES action, thereby contributing to the highernuclear accumulation of VP3 in SAOS-2 compared with SR40cells. Consistent with this, the nonphosphorylatable A108-substituted VP3 derivative showed significantly (P = 0.032)reduced nuclear accumulation in SAOS-2 cells compared withthe wild-type protein (Fig. 2B), implying that the lack ofphosphorylation allowed the NES to be active, resulting inreduced nuclear accumulation. That leptomycin B was able toincrease nuclear accumulation of this derivative significantly(P < 0.0001) in SAOS-2 cells (Fig. 2B), in contrast to the wild-type protein, was further consistent with this idea. T108 thusseems to inhibit NES action specifically in tumor as opposed tonormal cells, presumably through the fact that it is phosphor-ylated exclusively in tumor but not in normal cells (6).VP3 localizes in promyelocytic leukemia nuclear bodies

dependent on its NH2-terminal leucine-rich sequence. Asevident in the CLSM images in Figs. 1 and 2, GFP-VP3(1-121)localizes in distinct substructures within the nucleus, whichresemble PML nuclear bodies, whereas GFP-VP3(LRSm) showsnucleoplasmic localization (Fig. 2), implying that the LRSmutation impairs association with these substructures. Toconfirm this, immunostaining for PML protein, a predominantlyPML nuclear body–localizing protein, was done on GFP-VP3(1-121)–expressing SAOS-2/SR40 cells (Fig. 3A), the yellow colorationin the merge images (rightmost) confirming colocalization of VP3with endogenous PML. To further these observations, SAOS-2 cellswere cotransfected with plasmids encoding GFP-PML and redfluorescent protein (DsRed)-VP3(1-121), DsRed-VP3(74-121), orDsRed-VP3(LRSm), results indicating not only colocalization ofDsRed-VP3(1-121) with GFP-PML protein but also relocalization tothe cytoplasm of coassociated VP3 and PML proteins in cellsoverexpressing both (Fig. 3B). Similar results were obtained forDsRed-VP3(1-121) and a GFP fusion of the PML nuclear body–localizing homeodomain-interacting protein kinase HIPK2 (Fig. 3C ;ref. 16). DsRed-VP3(LRSm), in contrast to DsRed-VP3(1-121),showed no colocalization with GFP-PML within the nucleus, andno redistribution of either to the cytoplasm (Fig. 3B), implying thatthe LRS mutation had eliminated VP3 association with PML-containing complexes, and therefore that the wild-type LRSsequence is responsible for this association. Consistent with thisidea, cotransfection experiments using DsRed-VP3(74-121), whichlacks the LRS, revealed no colocalization with or cytoplasmicredistribution of PML (Fig. 3B). Intriguingly, DsRed-VP3(LRSm)retained the ability to colocalize with cotransfected GFP-HIPK2within the nucleus but not within PML nuclear bodies (Fig. 3C);thus, mutation of the LRS seemed to impair PML but not HIPK2association, implying that LRS-conferred VP3 association with

Tumor Cell–Specific Nuclear Targeting of Apoptin




PML nuclear bodies may be through direct binding to PML protein.These observations may relate integrally to the VP3 apoptoticmechanism in view of the fact that PML nuclear bodies, as well asPML and HIPK2 themselves, are linked to apoptosis (16).

Discussion

The present study uses quantitative approaches and isogenictumor/nontumor cell pairs to show for the first time that tumorcell–specific nuclear targeting on the part of VP3 is determined bya set of unique targeting signals. These comprise a bipartite-typetumor cell–specific nuclear targeting signal (NLS1 and NLS2), aphosphorylation-inhibited CRM1-recognized NES within the tumorcell–specific nuclear targeting signal, and a LRS (amino acids33-46) conferring interaction with PML nuclear body components(see schematic in Fig. 4). The most important finding is thatinhibition of nuclear export in tumor cells contributes critically to

the ability of VP3 to localize strongly in the nucleus of tumor but

not of normal cells. We show for the first time here that the

responsible NES lies within amino acids 97 to 105 and that it is

recognized by CRM1. We also show that the NH2-terminal LRS,

previously speculated to be a NES (1, 17), actually contributes to

nuclear accumulation rather than to cytoplasmic retention ornuclear export, as clearly indicated by the fact that its absence [e.g.,the GFP-VP3(74-121) construct] or inactivation by point mutation[the GFP-VP3(LRSm) construct] markedly reduces, rather thanincreases, tumor cell–specific nuclear targeting signal–dependentnuclear accumulation. The speculation that VP3 amino acids 33to 46 represent a cytoplasmic retention sequence (18), based onqualitative analysis in primary cells of a small set of VP3 truncationmutants, is not consistent with our quantitative analysis here,particularly of LRS and NES point mutant derivatives in the contextof full-length VP3. Our results show that the LRS facilitates nuclear

Figure 2. Nuclear accumulation of VP3is modulated by a COOH-terminalCRM1-recognized NES. A, CLSM imagesof SAOS-2 cells (top ) and SR40 cells(bottom ) 16 hours posttreatment toexpress the indicated GFP fusionconstructs in the absence (top ) andpresence of 2.8 Ag/mL leptomycin B (LMB ;added 5 hours before imaging) asindicated. B, quantitative analysis of thelevels of nuclear accumulation from CLSMimages such as those in (A) for SAOS-2cells (left) and SR40 cells (right ). Columns,mean Fn/c (n z 30); bars, SE. Significantdifferences (P values) for the Fn/cvalues between cells treated with/withoutleptomycin B are indicated.

Cancer Research




accumulation of VP3, with the activity, however, strongly depend-ent on the presence of NLS1 and NLS2.Another important finding is that VP3 NES activity is inhibited

by negative charge at T108. The A108 derivative VP3, lacking theability to be phosphorylated at T108, shows nuclear export activityin tumor cells comparable to that observed in normal cellsexpressing wild-type VP3, whereas the E108 derivative showsresistance to nuclear export even in normal cells, comparable tothe behavior of wild-type VP3 in tumor cells. Clearly, the action ofT108 is specifically to inhibit nuclear export, in contrast to previousspeculations (6) that the role of tumor cell–specific phosphoryla-tion at T108 may be to enhance VP3 nuclear import. The data here

clearly show that a critical contribution of T108 phosphorylation isto inhibit nuclear export, representing the mechanism of the tumorcell–specific activity of the NES activity (Fig. 4), whereas the basisof tumor cell–specific activity on the part of the tumor cell–specificnuclear targeting signal seems unclear. That specific phosphory-lation can inhibit/modulate NLS/NES activity has been shown for avariety of proteins (see ref. 19); the unique aspect with respect toVP3 is that the key phosphorylation event seems to be tumor cellspecific (see ref. 6). HIPK2, shown here to associate with VP3(Fig. 3C), would seem to be of prime interest in this regard becausepreliminary bioinformatic analysis (20) indicates that it is likely tophosphorylate VP3 at T108, supporting the idea that it may be the

Figure 3. Localization of VP3 with PML nuclear bodies is dependent on an NH2-terminal leucine-rich sequence. A, immunostaining for endogenous PML inuntransfected, fixed SAOS-2 and SR40 cells (left ) or cells transfected to express GFP-VP3(1-121) (right ). Staining was done using specific anti-PML primary (Santa Cruz)and Alexa 546–coupled secondary antibodies. Colocalization of endogenous PML transfected with GFP-VP3(1-121) is indicated by yellow coloration (Merge ). B, CLSMimages are shown of SAOS-2 cells transfected to express GFP-PML, DsRed-VP3(1-121), DsRed-VP3(74-121), and DsRed-VP3(LRSm) individually (top rows ), ortogether (lower rows ), as indicated. Cells were imaged live; colocalization of transfected GFP-PML and DsRed-VP3 is indicated by yellow coloration (Merge ). C, CLSMimages are shown of SAOS-2 cells transfected to express GFP-HIPK2, DsRed-VP3(1-121), and DsRed-VP3(LRSm) individually (top rows ), or together (lower rows ),as indicated. Cells were imaged live; colocalization of transfected GFP-HIPK2 and DsRed-VP3 is indicated by yellow coloration (Merge ).

Tumor Cell–Specific Nuclear Targeting of Apoptin




kinase responsible for the inhibition of VP3 nuclear export intumor cells. As the focus of future studies in this laboratory, acritical first step in testing this possibility will be to determinerelative levels of HIPK2 kinase activity in isogenic tumor/normalcell pairs, as well as in tumor tissues (6).

Acknowledgments

Received 4/20/2005; revised 6/13/2005; accepted 6/23/2005.

Grant support: Anti-Cancer Council of Victoria (Australia) and the AustralianNational Health and Medical Research Council ( fellowship 143790/334013 and projectgrant 143710).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank M. Yoshida (Chemical Genetics Laboratory, Discovery Research Institute,Riken, Wako, Saitama, Japan) for the gift of leptomycin B, and H.J. Lipps (Institutfur Zellbiologie, Universitat Witten/Herdecke, Witten, Germany) and A. Thorburn(Department of Cancer Biology and Comprehensive Cancer Center, Wake ForestUniversity School of Medicine, Winston-Salem, NC) for the pEPI and pEGFP-PML-C3/pEGFP-HIPK2-C3 plasmids, respectively.

Figure 4. Schematic model for differential regulation ofVP3 nuclear targeting in tumor and normal cells. VP3 istransported into the nucleus via the action of NLS1 andNLS2, most likely through the action of the importin h1protein (data not shown). In the nucleus, VP3 localizesprimarily to the PML nuclear body through the LRSsequence, possibly through direct interaction with PMLprotein itself. From these structures, unphosphorylatedVP3 is recognized by CRM1 through the COOH-terminalNES resulting in export into the cytoplasm in normal cells;in tumor cells, phosphorylation at T108 prevents NESaction, inhibiting nuclear export and resulting in increasednuclear accumulation. That LRS-mediated PML nuclearbody targeting precedes and/or may be a prerequisite forCRM1-mediated nuclear export is implied by the fact thatin the absence of the functional LRS [in GFP-VP3(74-121)or GFP-VP3(LRSm)], leptomycin B treatment does notincrease nuclear accumulation in normal cells when exportis blocked through leptomycin B (see Fig. 2).

Cancer Research


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2005;65:7059-7064. Cancer Res Ivan K.H. Poon, Cristina Oro, Manisha M. Dias, et al. Normal but not in Tumor CellsCRM1-Recognized Nuclear Export Signal that Is Active in Apoptin Nuclear Accumulation Is Modulated by a

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