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Journal of Virology, January 2005, p. 1339-1341, Vol. 79, No. 2
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.2.1339-1341.2005

LETTER TO THE EDITOR

A Tumor Cell-Specific Nuclear Targeting Signal within Chicken Anemia Virus VP3/Apoptin


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LETTER
 
Cancer is a growing problem for human health worldwide, with a dearth of efficient and above all specific anticancer treatments. Viral protein 3 (VP3; also known as apoptin), a gene product from the chicken anemia virus, represents a potential novel anticancer tool. It appears to have innate tumor-specific p53-independent, Bcl-2-enhanced proapoptotic activity and hence is of considerable interest to achieve efficient targeting and specific elimination of cancer cells (1, 2, 7, 10, 11). Intriguingly, the antitumor cell proapoptotic activity of VP3 appears to be integrally linked to its ability to localize in the nuclei of transformed cells, but not in those of primary or nontransformed cells (2, 7).

Claims as to VP3's tumor cell-specific nuclear targeting and proapoptotic activities are based thus far on analysis with nonisogenic cell pairs (7). Noteborn and colleagues in particular have performed a number of studies comparing VP3's properties in transformed and nontransformed cells of diverse types and origins (e.g., SAOS-2 human osteosarcoma cells compared with VH10 normal human skin fibroblasts) (1, 2, 10, 11), making it difficult to conclude that the apparent differential properties of VP3 in tumor or transformed cells are attributable to their tumorigenic status rather than to any number of other differences between the cell types or species, etc., used.

A recent study by Wadia et al. (9) reported that VP3 contains a "concentration-dependent" nuclear targeting signal (nuclear localization sequence [NLS]), rather than a "tumorigenic selective NLS" based on a comparison of ras-transformed mouse 3T3 cells and primary human fibroblasts. Transfection experiments carried out with increasing amounts of DNA led to the conclusion that better expression in tumor cells enabled greater nuclear accumulation, based on arbitrary scoring of microscopic images (9).

Claims as to VP3's tumor-specific nuclear targeting ability or otherwise need to be based on quantitative analyses of isogenic cell pairs at the single-cell level, where cell-cell variation in terms of actual expression can be measured rather than assumed according to the amount of transfecting DNA added. As a first step toward this goal, we have analyzed VP3's nuclear targeting abilities in two different isogenic cell pairs, thus enabling definitive comparisons between its activity in cells identical in genotype except for their transformed or nontransformed status. The lines used were (i) CV-1 African green monkey kidney cells (nontransformed) together with the simian virus 40-transformed derivative COS-7 line (3) and (ii) the tumorigenic SAOS-2 cell line, mutated in the retinoblastoma (Rb) tumor suppressor gene product, together with the nontransformed SR40 counterpart derived by transfection of SAOS-2 with the full-length Rb cDNA (4, 6).

Cells were transfected to express green fluorescent protein (GFP) or the VP3-GFP fusion constructs shown in Fig. 1A and imaged live 16 h later by confocal laser scanning microscopy (CLSM). In contrast to previous reports (1, 2, 10, 11), but consistent with the report of Wadia et al. (9), full-length VP3 was found to confer nuclear localization of GFP in both nontransformed cell types (CV-1 and SR40), as well as in the corresponding isogenic COS-7 and SAOS-2 transformed lines (Fig. 1B). The transformed lines, however, accumulated GFP-VP3(1-121) to a two times greater extent than did their nontransformed counterparts (P < 0.002), as revealed by determination of the nuclear-to-cytoplasmic ratio (Fn/c), with GFP-VP3(1-121) accumulating to levels about 70- and 50-fold greater than those in the cytoplasm of COS-7 and SAOS-2 cells, respectively (Fig. 1C). Thus, VP3 localizes to a greater extent in the nuclei of transformed compared to nontransformed cells.



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  		FIG. 1. Tumor cell-specific nuclear accumulation of VP3. (A) GFP-VP3 fusion proteins expressed from transfection constructs used in this study. (B) CLSM images of SAOS-2 and SR40 cells 16 h after transfection (with 1 µg of plasmid DNA per 6 x 105 cells and the Mirus TransitIT-LT1 transfection agent) to express the indicated GFP fusion constructs. (C) Quantitative analysis of the levels of nuclear accumulation (Fn/c after subtraction of background fluorescence), as determined by image analysis with the Image J public-domain software as previously described (5), from CLSM images such as those in panel B for SAOS-2 and SR40 cells and COS-7 and CV-1 cells. Results are the mean ± the standard error of the mean [n = ≥43 for SAOS-2 and SR40 cells and n = ≥20 for COS-7 and CV-1 cells for all constructs except GFP-VP3(1-73), where n = ≥7 and n = ≥15 for SAOS-2 and SR40 cells and COS-7 and CV-1 cells, respectively]. Statistically significant differences (Student's t test) between transformed and nontransformed isogenic cells are indicated. (D) Analysis of the levels of nuclear accumulation for GFP-VP3(1-121) (left) and GFP-VP3(74-121) (right) in low (Fn = <20)-, medium (Fn = 20 to 40)-, and high (Fn = >40)-expression SAOS-2 and SR40 cells (data from panel C). Results are the mean ± the standard error of the mean. NES, nuclear export signal.

The key nuclear targeting determinant was localized to the VP3 C terminus, consistent with the results of Danen-van Oorschot et al. (2) and Wadia et al. (9), whereby the 73 N-terminal residues of the 121-amino-acid VP3 protein conferred only cytoplasmic localization on GFP, in contrast to amino acids 74 to 121 (Fig. 1B and C). Quantitative analysis indicated, strikingly, that the latter possessed tumor cell-specific nuclear targeting ability; GFP-VP3(74-121) localized strongly in the nuclei of COS-7 and SAOS-2 cells (to levels about 20-fold greater than in the cytoplasm), in marked contrast (P < 0.0005) to their nontransformed isogenic counterparts (Fn/c values of ca. 4 and 11, respectively). VP3 residues 74 to 121 thus harbor an NLS that is highly efficient in transformed but not in nontransformed cells; this is presumably the basis of full-length VP3's ability to accumulate much more efficiently in tumor cells than in normal cells (1, 2).

To test whether activity of the tumor cell-specific NLS was responsive to the cellular expression level, as postulated by Wadia et al. (9), single-cell results for SAOS-2 and SR40 cells were grouped into low-, medium-, and high-expression cells on the basis of the VP3 expression level as determined by image analysis (Fig. 1D). No relationship was apparent between the level of expression and the extent of nuclear accumulation of GFP-VP3(1-121) (left panel) and GFP-VP3(74-121) (right panel) in either transformed SAOS-2 cells or nontransformed SR40 cells. Rather than high expression resulting in increased nuclear accumulation, the trend in the case of GFP-VP3(74-121) in SAOS-2 cells at least was that increasing levels of expression correlated with reduced nuclear accumulation (Fig. 1D).

On the basis of this quantitative analysis of isogenic transformed-nontransformed cell pairs, we conclude that chicken anemia virus VP3 possesses a specialized NLS (within residues 74 to 121) that shows high specificity for transformed cells. The only nuclear targeting signal with comparable specificity for a particular type of cell is the 45-amino-acid "developmentally regulated" NLS from adenovirus E1a protein (8), which functions in nuclear targeting only early (up to the early neurula stage) in Xenopus embryonic development. Future studies should establish the mechanistic basis of the action of the VP3 tumor cell-specific NLS in transformed cells as opposed to normal cells.


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FOOTNOTES
 
{dagger} Center for Developmental Biology
Institute of Genetics and Developmental Biology,Chinese Academy of Sciences
Beijing, Peoples Republic of China Back


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  6. Kaelin, W. G., Jr., D. C. Pallas, J. A. DeCaprio, F. J. Kaye, and D. M. Livingston. 1991. Identification of cellular proteins that can interact specifically with the T/E1A-binding region of the retinoblastoma gene product. Cell 64:521-532.[CrossRef][Medline]
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I. K. H. Poon
C. Oro
M. M. Dias
J.-P. Zhang{dagger}
D. A. Jans*
Nuclear Signaling Laboratory
Department of Biochemistry and Molecular Biology
Monash University, P.O. Box 13D
Monash, Victoria 3800, Australia

* Phone: 00613/99053778, Fax: 00613/99054699, E-mail: David.Jans{at}med.monash.edu.au

Journal of Virology, January 2005, p. 1339-1341, Vol. 79, No. 2
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.2.1339-1341.2005




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  • Maddika, S., Booy, E. P., Johar, D., Gibson, S. B., Ghavami, S., Los, M. (2005). Cancer-specific toxicity of apoptin is independent of death receptors but involves the loss of mitochondrial membrane potential and the release of mitochondrial cell-death mediators by a Nur77-dependent pathway. J. Cell Sci. 118: 4485-4493 [Abstract] [Full Text]  

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