Kooistra, K.
Citation
Kooistra, K. (2007, October 11). Viral elements inducing tumor-related apoptosis. Leiden
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V IRAL APOPTOSIS INDUCER APOPTIN SENSES
CELL - TYPE DEPENDENT NUCLEAR
TRAFFICKING OF THE TRANSFORMING
SV40 LARGE T ANTIGEN
Klaas Kooistra1, Dongjun Peng1, Claude Backendorf2, Mathieu H.M.
Noteborn1 and Ying-Hui Zhang2
1Biological Chemistry, Leiden Institute of Chemistry, 2333 CC Leiden
2Molecular Genetics, Leiden Institute of Chemistry, 2333 CC Leiden
ABSTRACT
Simian virus 40 (SV40) large tumour antigen (LT) targets the nuclear matrix as part of its transforming effect. Here we report that in microinjection experiments the transforming protein SV40 LT rapidly enters the nucleus of human normal VH10 fibroblasts, whereas its nuclear targeting is significantly delayed in human primary mesenchymal stem cells (hMSC). As this differential behaviour might reflect the protein's differential transforming activity in these human cell types, we examined the effect of SV40 LT nuclear trafficking on the activation of the transformation-specific apoptosis inducer apoptin, a viral protein derived from chicken anaemia virus. In VH10 cells where SV40 LT is swiftly and extensively targeted to the nucleus, co- expressed apoptin was instantly activated to translocate to the nucleus and subsequently induced apoptosis. In hMSC cells, where delayed nuclear trafficking of SV40 LT was observed, apoptin maintained its normal-specific properties characterized by aggregation, epitope-shielding, and/or degradation in the cytoplasm, resulting in a significantly slowed down activation. At later time points, when SV40 LT appeared in the nucleus of hMSC cells, apoptin's assumed its transformation-specific form, resulting in its nuclear accumulation and apoptosis induction. These results indicate that SV40 LT, in a cell-type dependent manner, triggers transformation-related processes in the nucleus, which are particularly sensed by apoptin.
Keywords: apoptin, SV40 large T antigen, nuclear trafficking, cellular transformation, transformation-specific apoptosis
INTRODUCTION
Viral oncogenes play central roles in reprogramming infected cells to a state of constitutive proliferation (Munqer et al., 2006). More than 15% of human cancers have a viral etiology (Shera et al., 2001). The oncogenic DNA virus simian virus 40 (SV40) has served as a model for understanding cell transformation (Barbanti-Brodano et al., 2004; Ahuja et al., 2005). Recent studies have linked SV40 to cancer such as non- Hodgkin's lymphoma, especially in countries where the population was exposed to SV40- contaminated polio vaccines prior to 1963 (Chen et al., 2006), suggesting the involvement of SV40 in tumorigenesis. The major oncoprotein large tumour antigen (LT) encoded by SV40 has been widely used to convert various cell types to a transformed and immortal phenotype, and also to induce progressive tumours in e.g.
transgenic animals (Hahn et al., 2002; Kirchhoff et al., 2004). SV40 LT corrupts the cellular checkpoint control mechanisms that guard cell division and the transcription, replication and repair of DNA (Welcker & Clurman, 2005; Ahuja et al.,
2005). To achieve these derailing transforming activities, SV40 LT has to integrate the nuclear matrix of the targeted cells (Xiao et al., 1997; Deppert, 2001).
The chicken anaemia virus derived protein apoptin has been shown to sense cell- transformation-related processes (Noteborn, 2004; Rohn & Noteborn, 2004; Heilman et al., 2006; Alvisi et al., 2006; Maddika et al., 2006). In transformed and/or cancerous cellular states, apoptin becomes phosphorylated, translocates to the nucleus and induces apoptosis (Danen-Van Oorschot et al., 1997; Rohn et al., 2002). In normal cells, apoptin is primarily present in the cytoplasm and becomes epitope-shielded and is eventually degraded (Danen-Van Oorschot et al., 1997; Zhang et al., 2003). In normal human fibroblasts apoptin senses the transient transforming signals transmitted by ectopically expressed SV40 LT (Zhang et al., 2004). Upon expression of SV40 LT, apoptin becomes rapidly phosphorylated, translocates from the cytoplasm to the nucleus and induces apoptosis. SV40 LT that lacks a nuclear localization signal is unable to activate apoptin's activities; apoptin can not translocate to the nucleus and can not induce apoptosis (Zhang, 2004; Zhang et al., 2004).
Here, we report that after DNA-microinjection, the transforming protein SV40 LT translocates immediately to the nucleus of human normal VH10 fibroblasts but displays a clearly delayed nuclear trafficking in human primary mesenchymal stem cells (hMSC). This delay interferes with the protein's transforming activity, as sensed by the transformation-specific apoptosis inducer apoptin.
METHODS
Cells and cell culture.
Human diploid skin fibroblasts (VH10) derived from a healthy individual (Klein et al., 1990) and the human osteosarcoma cell line Saos-2 (Zhang et al., 2003) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal calf serum, 100 U/ml penicillin, and 100 ȝg/ml streptomycin (Life Technologies). Human bone-marrow- derived mesenchymal stem cells (hMSC; Cat. # PT-2501, LOT# 9F1938, BioWhittaker Europe SPRL, Belgium) were recovered and cultured following delivery at passage 1 in a T-25 flask with the accompanying medium MSCGM Bullet-kit (PT-3001, BioWhittaker), and used for microinjection experiments from passage 2 to passage 5 as recommended by the supplier.
DNA plasmids.
The DNA plasmids pCMV-VP3 encoding apoptin and pCMV-Desmin, expressing the neutral non-apoptosis inducing protein desmin, have been described previously
(Noteborn et al. 1994; Danen-Van Oorschot et al., 1997). In pRSV-LT (formerly 100 named pR-S884), a complete SV40 LT together with a 62 amino-acid C-terminal truncated small T (st) protein was expressed under the RSV promoter. The plasmid was created by substituting the 1169 bp HindIII fragment of pRSV-SV40 encompassing the st coding region with the corresponding 922 bp HindIII fragment of pR-SV40-dl884 with a deletion in the C-terminus of st (Gorman et al., 1983; De Ronde et al., 1998).
Plasmid pcDNA3-HA-FADD, expressing the apoptosis inducing protein FADD (Chinnaiyan et al., 1995), was kindly provided by Prof. Dr. Vishva Dixit (University of Michigan, USA).
Microinjection of DNA.
DNA microinjection into adherent cells was performed on a workstation equipped with a menu-controlled programmable micromanipulator InjectMan NI 2 mounted on a Zeiss-microscope Axiovert 200 and a microinjector FemtoJet (Eppendorf).
Microinjection needles with a 0.5 ± 0.2 ȝm diameter tip (Sterile Femtotips II, Eppendorf) were used for microinjection and in general loaded with 50 ȝl of each injection sample by use of Eppendorf microloaders. DNA delivery into the nucleus of cells was carried out under an injection pressure of 40-50 hPa and an injection time of 0.2-0.5 sec. The concentration of DNA for microinjection was determined at 50 ng/ȝl.
To trace injected cells, when needed, the injection solution was supplemented with 1 mg/ml dextran-conjugated tetramethylrhodamine (Rho-Dex; 70,000 MW, lysine fixable; Molecular Probes). Prior to microinjection, all injection samples were microcentrifuged at 13,000 rpm (Centrifuge 5415 R, Eppendorf) at 4°C for 15 min to eliminate any precipitates that might clog the microinjection needles. Twenty-four hours prior to microinjection, cells were seeded at 30-40% confluence in 35-125 mm tissue culture dishes with a glass-bottomed microwell (P35G-1.5-14-C, No. 1.5, uncoated, MatTek Corporation, Ashland, MA). During microinjection, cells were incubated in RPMI1640 medium (without phenol red, containing 25 mM HEPES pH 7.2, 5% foetal calf serum, penicillin, and streptomycin; Life Technologies). For each DNA micro- injection experiment, at least 100 cells were injected within short time, not longer than15 minute exposure to the air. Immediately after microinjection, cells were given fresh culture medium and incubated at 37°C and 5% CO2.
Indirect immunofluorescence assay.
After microinjection, cells were fixed at a given time with 1% formaldehyde (freshly made in PBS) for 10 min, cold methanol for 5 min, and then cold 80% acetone (in H2O) for 2 min. The expression and cellular localization of proteins encoded by injected DNA plasmids were studied by indirect immunofluorescence as described previously (Danen-Van Oorschot et al., 1997; Zhang et al., 2004). To detect the presence and cel-
lular localization of ectopically expressed proteins in injected cells, several antibodies were selected. Apoptin was visualized by the apoptin-specific rabbit antibody RĮVP3C, which recognizes an epitope residing between amino acids 76-90 in the C-terminus of apoptin (Zhang et al., 2004). The mouse monoclonal antibody 419 (Research Diagnostic Inc.), which is reactive with the first 82 N-terminal amino acids of SV40 LT, was used to determine SV40 LT protein. In certain experiments, the mouse monoclonal antibody against human FADD (BD Transduction Laboratories, Belgium) was used to probe cells transduced with the construct pcDNA3-HA-FADD as a positive control for apoptosis induction. The apoptotic negative control protein desmin was detected by mouse monoclonal antibody mAb 33 (Monosan, Uden, the Netherlands), as described previously (Danen-van Oorschot et al., 1997). The appropriate fluorescein- isothiocyanate (FITC)- or rhodamine-isothiocyanate (Rho)-labelled goat antibodies (Jackson ImmunoResearch Laboratories, West Grove, Pa.) were used as secondary antibodies. The nuclear morphology indicative of an apoptotic state was determined by DAPI (2,4-diamidino-2-phenylindole) staining of nuclear DNA (Danen- Van Oorschot et al., 2000). Immunofluorescence stained cells were analyzed by fluorescence microscopy (Olympus, PROVIS AX). Images were captured by digital image analysis equipment (Sony DXC-950P, 3CCD colour video camera) and processed with analysis® software (3.00, Soft Imaging System).
RESULTS
Differential nuclear trafficking of SV40 LT in human normal cell types.
Nuclear targeting is an intrinsic and functionally relevant property of SV40 LT (Deppert, 2000). To address the nuclear trafficking behaviour of SV40 LT in human normal cells, we selected two cell types, VH10 fibroblasts and hMSC mesenchymal stem cells, and microinjected both cell types with plasmid DNA pRSV-LT expressing SV40 LT. At regular time points, the cells were fixed and analyzed for SV40 LT expression and its cellular localization by means of indirect immunofluorescence assay.
Figure 1 shows representative expression and subcellular distribution of SV40 LT in microinjected VH10 and hMSC cells, respectively at 6, 24 and 48 hours post microinjection (PM). In VH10 cells, SV40 LT was completely gathered in the nucleus already at 6 hours PM, retaining its constant nuclear targeting with the time course (Fig.
1a, left panel; Fig 1b, bottom graph). In hMSC cells, however, LT protein was detected almost solely in the cytoplasm at 24 hours PM (Fig. 1a, right panel). Forty-eight hours PM, LT protein was present in both cytoplasm and nucleus of hMSC cells, and a relative small proportion of LT-positive hMSC cells harboured LT selectively in their nucleus (Fig. 1a, right panel; Fig. 1b, upper graph). These results reveal that SV40 LT
protein was imported into the nucleus of human VH10 fibroblasts much more swiftly than in hMSC cells. Therefore, we conclude that nuclear trafficking of SV40 LT can vary depending on the cellular background.
Figure 1. Differential nuclear trafficking of the transforming SV40 large T antigen (LT) in human fibroblasts (VH10) versus human mesenchymal stem cells (hMSC). (a) Distinct subcellular location of SV40 LT in VH10 and hMSC cells. The cells were nuclearly microinjected with plasmid DNA pRSV-LT encoding LT, fixed at 6, 24 or 48 hours post microinjection (PM), and stained with the LT- specific mouse monoclonal antibody 419 by indirect immunofluorescence assay. Fluorescence microscopy images are shown of representative cells indicating LT subcellular localization. Image original magnification: x1000. (b) Differential dynamics of SV40 LT nuclear trafficking in VH10 versus hMSC cells. Solid black bar: exclusive nuclear localization; solid grey bar: nuclear and cytoplasmic localization; open bar: only cytoplasmic localization. Two independent experiments were carried out. Per given time point at least 100 positive cells were scored.
Apoptin displays its normal-cell-specific properties in human primary mesenchymal stem cells.
Nuclear localization of SV40 LT is known to be required for induction and maintenance of transformation-related processes in human cells (Deppert, 2000; Lee & Langhoff, 2006). Therefore, we sought to address whether the differential nuclear trafficking of LT observed in VH10 versus hMCS cells is relevant for induction of transformation- related processes. Our readout method was established on the basis of the cell- transformation-specific apoptosis-inducing protein apoptin, as apoptin can be activated to translocate to the nucleus and induce apoptosis in stably-transformed and cancerous cells (Danen-Van Oorschot et al., 1997; Rohn et al., 2002) as well as in transiently- transformed human fibroblasts showing nuclear SV40 LT (Zhang et al., 2004).
First, to confirm how apoptin would behave by itself in hMSC cells, the plasmid DNA pCMV-VP3 encoding apoptin protein was transduced into the nucleus of hMSC cells by microinjection. As a control for characteristic apoptosis induced by apoptin, human transformed osteosarcoma Saos-2 cells were microinjected also with pCMV- VP3 plasmid. Meanwhile, the plasmid pCMV-Des encoding desmin was used as a negative control for apoptosis induction. As expected, apoptin expressed by microinjected pCMV-VP3 was predominantly present in the nucleus of the transformed Saos-2 cells as early as 6 hours PM (Fig. 2a, left panel) when approximately 20% of the cells were apoptotic (Fig. 2b). After 24 and 48 hours, respectively 50, 70% of the apoptin- expressing underwent apoptosis (Fig. 2a, right panel; Fig. 2b), confirming the cell- transformation-specific apoptotic activity of apoptin. On the contrary, apoptin protein was mainly localized in the cytoplasm of hMSC cells and did not induce obvious apoptosis, similar to the apoptosis- negative control protein desmin (Fig. 2c; Fig. 2d). To exclude the possibility that the resistance of hMSC cells to apoptin could be due to a general resistance to apoptosis, hMSC cells were nuclearly microinjected with a plasmid encoding the cellular protein FADD that is known to induce apoptosis in a broad range of human normal and transformed cells. Clearly, many FADD-expressing hMSC cells were subject to FADD-induced apoptosis, even as early as 6 hours PM (30%) (Fig. 2c; Fig. 2d).
In addition, we examined whether after DNA microinjection in hMSC cells apoptin would behave in a normal-cell-specific fashion as observed previously (Zhang et al., 2003).
To that end, the fluorescent marker Rho-Dex was co-microinjected with pCMV-VP3 into the nucleus of hMSC cells to trace the injected cells. In the Rho-Dex positive hMSC cells, apoptin was distributed as fine particles in the cytoplasm at early time (6h, Fig. 3), followed by gradual aggregation in the cytoplasm (<24h & >24h, Fig. 3) and final epitope-shielding or degradation (72h, Fig. 3) at later time points. These observations are in complete agreement with those made by microinjection of the
Figure 2. Cellular transformation-related status determines apoptin's nuclear localization and apoptotic activity. Human transformed osteosarcoma Saos-2 cells were microinjected into the nucleus with the plasmid pCMV-VP3 encoding apoptin. Human primary mesenchymal stem cells (hMSC) were nuclearly microinjected with the same pCMV-VP3, the plasmid pCMV-Des encoding non- apoptotic protein desmin (negative control), or the plasmid encoding apoptotic protein FADD (positive control), respectively. The microinjected cells were fixed at the indicated time, and stained with apoptin-specific rabbit antibody RĮVP3C, mouse monoclonal antibody mAb33 against desmin, or mouse monoclonal antibody against FADD to detect the corresponding proteins by means of indirect immunofluorescence assay. Nuclear DNA was stained with DAPI to show the fate of the cells in the presence of relative proteins. At least 100 cells per given time point were analyzed in two independent experiments. Error bars indicate standard deviation. Fluorescence microscopy images are shown for representative cells. Image original magnification: x1000. (a) Nuclear localization and apoptotic activity of apoptin in human transformed Saos-2 cells. (b) Percentage of apoptosis induction by apoptin in transformed Saos-2 cells at given time PM. (c) Representative immunofluorescence microscopy images of human primary hMSC cells responding to apoptin, desmin (the negative apoptotic control), and FADD (the positive apoptotic control). (d) Comparative apoptosis induction (%) of hMSC cells by apoptin, desmin and FADD at given time PM.
Figure 3. Normal-specific behaviour of apoptin in the cytoplasm of human primary mesen- chymal stem cells (hMSC). hMSC cells were nuclearly co-microinjected with apoptin expressing plasmid pCMV-VP3 together with the fluorescent marker Rho-Dex, fixed at indicated time, and stained with apoptin-specific rabbit antibody RĮVP3C to visualize apoptin. In Rho-Dex traced cells, various appearances of apoptin in the cytoplasm are shown: fine particles (6h), gradual clustering (<24h), increased aggregation (>24h), and eventual epitope-shielding or degradation (48h). The bottom panel images represent the merged fluorescent microscopy images of apoptin (green), microinjection marker Rho-Dex (red), and DAPI-stained nuclear DNA (blue) of the identical injected cells. Image original magnification: x1000.
recombinant apoptin fusion protein MBP-apoptin in hMSC cells (Zhang et al., 2003).
In all cases, parallel DNA microinjection experiments were carried out in human normal VH10 fibroblasts. The results were as reported before (Danen-Van Oorschot et al., 1997; Zhang et al., 2003) and very similar to the present observations in hMSC cells (data not shown).
Nuclear trafficking of SV40 LT results in transformation-related events sensed by apoptin.
Next, we examined whether differential nuclear trafficking of SV40 LT would have an influence on its transforming activity, i.e. on the activation of apoptin's nuclear translocation and apoptotic activity. To that end, human VH10 fibroblasts and hMSC cells were co-microinjected into their nucleus with plasmids encoding apoptin and LT. At several time points PM, the cells were fixed and analyzed by means of fluorescence microscopy by monitoring the subcellular localization and apoptotic activity of apoptin. In parallel, the expression and localization of LT were analyzed.
Already at 6 hours PM, co-synthesis of LT and apoptin resulted in a nuclear and nuclear/cytoplasmic localization of apoptin in a significant amount of microinjected VH10 cells (Fig. 4a, top graph). In contrast, in apoptin and LT co-expressing hMSC cells, only a few cells showed apoptin distributed over both nucleus and cytoplasm. None of the LT/apoptin-positive hMSC cells could be observed to have apoptin exclusively in the nucleus (Fig. 4a, bottom graph). More than 90% of LT/apoptin-positive VH10 cells revealed nuclear/cytoplasmic localization of apoptin at 24 hours PM, and 35% could acquire nuclear-restricted apoptin at 48 hours PM (Fig. 4a, top graph). Significantly different, the majority of co- microinjected hMSC cells deposited apoptin in the cytoplasm, with only 28% having apoptin distributed over both nuclear and cytoplasmic compartments late at 48 hours PM (Fig. 4a, bottom).
By means of DAPI-staining (Danen-Van Oorschot et al., 2000), we also determined the level of apoptin-induced apoptosis in the co-microinjected LT/apoptin-positive cells. The results showed that apoptin induced apoptosis in VH10 cells with nuclear LT (Fig. 1a & 1b) to a level of 74% at 48 hours PM (Fig. 4b, top graph), as morphologically indicated by DAPI-staining (Fig. 4c, top panel). This figure was significantly higher than in hMSC cells where LT was retarded in the cytoplasm.
Yet, once a threshold amount of LT had gradually entered the nucleus of hMSC cells late at 48 hours PM (Fig. 1a & 1b), apoptin was activated to induce apoptosis (Fig. 4c, bottom panel) to a level of 26% (Fig. 4b, bottom graph).
The results above revealed that delayed nuclear trafficking of LT in human cells defers its transforming capacity, which can be sensed by the transformation-specific viral protein apoptin. When nuclear targeting of LT is delayed, apoptin's nuclear translocation is retarded and its apoptotic activity is sequentially reduced.
Cytoplasmic non-transforming SV40 LT allows apoptin's normal-cell-specific properties.
Apoptin protein becomes aggregated, epitope-shielded and finally degraded in the cytoplasm of various normal non-transformed cells as found before (Zhang et al., 2003) and as reported here for hMSC cells (Fig. 3). Therefore, we analyzed in closer detail the fate of apoptin protein in hMSC cells co-microinjected with LT and apoptin encoding plasmids. Figure 5 shows representative co-microinjected hMSC cells visualized by indirect immunofluorescence staining, indicative of the subcellular localization and structures of both apoptin and LT. Six hours PM, in almost all injected cells both apoptin and LT were situated in the cytoplasm, where apoptin was distributed in fine granular speckles (6h, Fig. 5). After 24 hours, while LT was still mainly located in the cytoplasm, apoptin was condensed into huge aggregates (24h, Fig. 5), which were visible until 48 hours PM. At this time point, although a few cells harboured LT in
Figure 4. Nuclear trafficking of SV40 LT enables apoptin-sensed transforming activity. Human normal fibroblasts (VH10) and primary mesenchymal stem cells (hMSC) were nuclearly co-microinjected with plasmids pRSV-LT encoding LT and pCMV-VP3 encoding apoptin and analyzed at 6, 24 and 48 hours PM for apoptin's subcellular localization and apoptosis induction. Two independent experiments were carried out. Per indicated time point at least 100 positive cells were scored. (a) Subcellular localization of apoptin. Solid black bar: exclusive nuclear localization; solid gray bar: nuclear and cytoplasmic localization; open bar: only cytoplasmic localization. (b) Percentage of apoptosis induction by apoptin at given time PM. (c) Representative immunofluorescence microscopy images of apoptotic VH10 and hMSC cells induced by apoptin at 48 hours PM. Apoptin expression was detected with apoptin-specific rabbit antibody RĮVP3C (green), the presence of LT with the mouse monoclonal antibody 419 against SV40 LT (red), and nuclear DNA with blue DAPI-staining (shown in
"merge"). Image original magnification: x1000.
the nucleus, apoptin remained as aggregates in the cytoplasm (48h, Fig. 5). At 72 hours, a significant amount of LT had entered the nucleus of the co-microinjected hMSC cells.
In some of these cells, no apoptin could be detected (72h, Fig. 5), displaying apoptin's
normal-cell-specific properties, namely, cytoplasmic epitope-shielding and/or degradation, thus explaining the observed reduction in apoptotic activity. In other cells, apoptin was activated and induced apoptosis (Fig. 4b, bottom graph; Fig. 4c, bottom panel).
These results demonstrated that cytoplasmic restriction of LT results in non-transformed conditions enabling apoptin to become aggregated in the cytoplasm, shielded and finally degraded in a normal-cell-specific fashion, and further confirming that nuclear translocation of LT is required for initiation of cell-transformation events, which apoptin can sense resulting in induction of apoptosis in a transformation-specific manner.
Figure 5. Normal-cell-specific neutralization of apoptin in the presence of cytoplasmic LT. Human primary mesenchymal stem cells (hMSC) were nuclearly co-microinjected with plasmids pCMV-VP3 encoding apoptin and pRSV-LT encoding LT. The cells were fixed after 6, 24, 48 and 72 hours PM and double- stained with the apoptin-specific rabbit antibody RĮVP3C (green) and the mouse monoclonal antibody 419 reactive with SV40 LT (red) by indirect immunofluorescence assay. Nuclear DNA was detected by DAPI-staining (blue). Fluorescence microscopy images, including merged images, are shown of representative cells. Image original magnification: x1000.
DISCUSSION
The present study shows that the dynamics of SV40 LT nuclear translocation is cell-type dependent. Delayed nuclear trafficking of LT interferes with its cell-transformation capabilities, which can be sensed by the transformation-specific apoptotic inducer apoptin.
Cytoplasmic LT correlates with normal-cell-specific aggregation, epitope-shielding and final degradation of apoptin, whereas nuclear LT stimulates transformation-specific activities of apoptin, such as its nuclear transfer and apoptosis induction.
Besides human mesenchymal stem cells (hMSC), mouse embryonic fibroblasts seem to exhibit a delayed nuclear trafficking of LT similarly (Pietersen & Zhang, unpublished observations). The observation that LT enters the nucleus in a cell-type specific manner may provide opportunities for understanding the cellular factors playing a role in the nuclear trafficking of transforming proteins such as LT. The nuclear matrix, as the key integrator of nuclear structure and function, is an important target for structural and functional alterations during the process of transformation. Such alterations have indeed been observed and even used as hallmarks for this process (Deppert, 2000). Studies using SV40 encoded proteins support the concept that the nuclear matrix can be targeted by viral and cellular oncogenic products and that this nuclear targeting contributes to cellular transformation and tumorigenesis (Holth et al., 1998; Samuel et al., 1997). Because SV40 LT specifically targets the chromatin/
nuclear scaffold during cellular transformation (Deppert, 2000), it is most likely that LT requires, at least in part, access to the nuclear matrix or other nuclear components to elicit the transformation-related responses. Human mesenchymal stem cells have the potential capacity for proliferation and a strong "stem cell plasticity" for differentiation into different tissues, including bone, cartilage and muscle, and even for transdifferentiation into skin, liver, neurons and glia brain cells (Krabbe et al., 2005; Vaananen, 2005; Catelas et al., 2006). However, more research is needed to reveal the molecular mechanisms of the hMSC potential for proliferation and differentiation (Vaananen, 2005). The delayed nuclear trafficking of LT in hMSC cells is perhaps due to differences in protein translocation control in this particular cell type. The fact that transformation-related processes induced by LT occur in a differential manner depending on the cell type situation, underlines once again that apoptin senses transformation-related nuclear processes (Poon et al., 2005).
Recently, studies with apoptin have revealed several distinct transformation-related processes (Rohn & Noteborn, 2004). One of these processes involves a transformation- related kinase activity, which is constitutively active in stably-transformed cells and tumour cells and is quickly activated in cells transiently expressing LT, but not in normal cells lacking LT (Rohn et al., 2002; Zhang et al., 2004; Poon et al., 2005).
The effect of delayed LT nuclear trafficking might result in a postponed triggering of the transformation-related kinase activity, a view which is strengthened by the fact that N-terminal LT sequences are only able to activate this specific kinase upon translocation to the nucleus in normal human VH10 fibroblasts (Zhang et al., 2004).
Our results indicate that apoptin in normal cells seems to encounter other cellular factors than in cancer cells. The mechanism of apoptin's cytoplasmic retention in normal cells is not fully understood. However, apoptin's normal-cell-specific properties of aggregation, shielding and final degradation in the cytoplasm may play a crucial role in preventing its apoptotic effect in normal cells, as shown in this study and
as recently reported (Zhang et al., 2003). Does nuclear transfer of LT prevent cytoplasmic retention of apoptin or is the post-translational modification of apoptin responsible for apoptin’s entry into the nucleus? Further work is needed to answer these questions.
ACKNOWLEDGEMENTS
We thank Dr. A.G. Jochemsen for kindly providing the SV40 protein expressing plasmid and B. Klein for VH10 cells. This work was partially supported by grants from the Royal Dutch Academy of Sciences and Arts and the Netherlands Ministry of Economic Affairs.
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