Viral elements inducing tumor-related apoptosis
Kooistra, K.
Citation
Kooistra, K. (2007, October 11). Viral elements inducing tumor-related apoptosis. Leiden
Institute of Chemistry (LIC), Faculty of Science, Leiden University. Retrieved from
https://hdl.handle.net/1887/12375
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in the
Institutional Repository of the University of Leiden
Downloaded from: https://hdl.handle.net/1887/12375
Note: To cite this publication please use the final published version (if applicable).
I NFLUENCE OF ABERRANT EXPERIMENTAL
CIRCUMSTANCES ON APOPTIN ’ S
“ NORMAL - CELL ” BEHAVIOUR
CHAPTER 5
Klaas Kooistra1, 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
Manuscript submitted
EXPERIMENTAL CIRCUMSTANCES AFFECTING APOPTIN’S BEHAVIOUR
105 Apoptin, a viral protein encoded by chicken anaemia virus, is characterized by its tumour- cell nuclear localization leading to apoptosis induction (Zhuang et al., 1995; Danen-van Oorschot et al., 1997; Danen-van Oorschot et al., 2003) and its normal-cell cytoplasmic retention leading to its degradation (Danen-van Oorschot et al., 1997; Zhang et al., 2003).
More than 70 human tumour-derived cell lines have been shown to be highly sensitive to apoptin-induced apoptosis (Rohn & Noteborn, 2004; Tavasolli et al., 2005). Apoptin- induced apoptosis in tumour cells is mediated by a tumour-related protein kinase activity (Rohn et al., 2002), a tumour-specific nuclear targeting signal within apoptin (Danen-van Oorschot et al., 2003; Poon et al., 2005a), and several apoptin associating proteins (Danen-van Oorschot et al., 2004; Teodoro et al., 2004; Heilman et al., 2005). The cytoplasmic retention of apoptin in normal cells has been suggested to be modulated by a CRM1-recognized nuclear export signal that is active in normal but not in tumour cells (Poon et al., 2005b). The view of apoptin as a tumour-specific death effector is indeed based on its cytosolic nontoxicity in human normal cells as opposed to its toxicity in tumour cells. We have noticed, however, that variations in experimental conditions may influence apoptin’s normal-cell-specific behaviour, for instance, increased nuclear translocation and apoptosis induction in normal cells, as was noted by several recent reports (Guelen et al., 2004; Wadia et al., 2004). Here, we briefly summarize our apoptin studies in normal cells of the past years performed under different experimental circumstances and communicate our experience shared with others who are interested in apoptin research.
In 2000, we established microinjection techniques especially to facilitate apoptin studies in human normal cells. Since then, a number of human primary cell types, including mesenchymal stem cells (MSC), hepatocytes (hNheps), and fibroblasts (VH10 or NHDF), have been tested for their responses to apoptin’s activity by microinjection of recombinant apoptin protein MBP-apoptin (Leliveld et al., 2003) or apoptin-expressing DNA plasmid pCMV-VP3 (Danen-van Oorschot et al., 1997). In these human normal cells, both protein and DNA microinjection studies confirmed apoptin’s classical normal-cell cytoplasmic pattern (Fig 1A & 1B). By means of both microinjection techniques, we demonstrated that the cytoplasmic apoptin becomes (epitope) shielded and eventually undergoes degradation (Zhang et al., 2003; Zhang, unpublished data). In more than 80 independent protein microinjection experiments and more than 40 individual DNA microinjection experiments, apoptin was consistently found to be diffusely dispersed in the cytoplasm of human normal cells and not toxic (Fig. 1A & 1B). Under the same circumstances, parallel microinjection experiments performed in human tumour Saos-2 cells showed predominant nuclear localization of apoptin and significant apoptosis induction (Leliveld et al., 2003;
Zhang et al., 2003; Kooistra et al., submitted). Hence, it is indicated that the differences in subcellular localization of apoptin and apoptotic effect in tumour versus normal cells are not due to different apoptin protein levels, at least in the case of our microinjection
CHAPTER 5
106
experiments. Therefore, the effects are not concentration-dependent as reported (Wadia et al., 2004).
Figure 1. Apoptin’s normal-cell behaviour under different experimental circumstances. (A) Cytoplasmic localization of recombinant apoptin protein MBP-apoptin (Leliveld et al., 2003) in different human normal cell types. (B) Cytoplasmic localization of apoptin expressed by nuclear microinjected DNA plasmid pCMV- VP3 (Danen-Van Oorschot et al., 1997) in the same human normal cell types. Human diploid foreskin fibroblasts VH10 (Danen-Van Oorschot et al., 1997) were cultured in supplemented DMEM and used at passage 11. Human bone-marrow derived mesenchymal stem cells (MSC) (Cat. # PT-2501, Lot. # 9F1938, BioWhittaker Europe SPRL, Belgium) were cultured with the accompanying medium MSCGM Bullet-kit (PT-3001, BioWhittaker) and used at passage 2. Human primary hepatocytes hNheps (Cat. # CC-2591, Lot.
# BioWhittaker Europe SPRL, Belgium) were seeded on an extracellular matrix (ECM, Sigma)-coated dish, cultured in the accompanying medium HCMTM (HCM Bullet-kit CC-3199+CC-4182, BioWhittaker), and used at passage 1. The concentration of protein and DNA plasmid used for microinjection experiments was determined at 3 mg/ml and 50 ng/μl, respectively. Microinjection was carried out under an injection pressure of 40-50 hPa and an injection time of 0.2-0.5 second with an equipped microinjection system (Eppendorf, Germany), as described previously (Zhang et al., 2003). (C) Two types of human fibroblasts VH10 and NHDF are examples of normal human fibroblasts that occasionally show apoptin’s nuclear translocation after DNA transfection. NHDF were CD31-negative normal human dermal fibroblasts, cultured in MEM-EARLE (BIOCHROM AG, Berlin) with relative supplements (Zhang et al., 2004), and used at passage 5. Apoptin either in recombinant protein form or expressed by DNA plasmid after microinjection or transfection was visualized by indirect immunofluorescence assay using a rabbit apoptin- specific antibody Į-VP3C (Zhang et al., 2003). Original image magnification: X 1000.
EXPERIMENTAL CIRCUMSTANCES AFFECTING APOPTIN’S BEHAVIOUR
107 In one specific DNA microinjection experiment, however, we observed that apoptin translocated into the nucleus of two types of human fibroblasts without causing cell death, whereas in parallel protein microinjection performed in the same cells still resulted in apoptin protein retention in the cytoplasm canonical (data not shown). In this case, the only changed parameter was the DNA plasmid batch. The quality of a DNA plasmid batch can differ depending on solvent, purity, bacteria source, etc. Hence, we conclude that the quality of apoptin-expressing DNA plasmid may affect apoptin’s normal-cell-specific behaviour.
The normal-cell cytoplasmic localization and nontoxicity of apoptin were first determined in various human normal diploid cells, such as human primary fibroblasts, keratinocytes, endothelial cells, smooth muscle cells and T cells, by use of DNA transduction methods, e.g. calcium-phosphate (Ca2PO4) precipitation transfection or N-[1-(2,3-dioleoyloxy)- N,N,N-trimethylammonium propane (DOTAP) transfection (Danen-van Oorschot et al., 1997). In these early experiments, both Ca2PO4 and DOTAP transfection methods were not sufficient to introduce apoptin into normal cells with high transfection efficiencies.
Subsequently, we used other DNA transfection methods, for instance, FuGene 6 Transfection Reagent, a multi-component lipid-based transfection reagent (Roche Applied Science), and Nucleofector AMAXATM (AMAXA Biosystems). With either FuGene or AMAXA transfection, we achieved high transfection efficiencies of apoptin in human normal fibroblasts, which enabled us to investigate apoptin’s regulation by a tumour- specific kinase activity (Rohn et al., 2002) and subsequently to reveal the activation of apoptin’s phosphorylation by transient transforming signals (Zhang et al., 2004).
Nevertheless, we noticed that nuclear translocation of apoptin in normal cells occurred after DNA transfection then after DNA microinjection, as revealed in two types of human normal fibroblasts VH10 and NHDF (Fig. 1C). Occasionally, an aberrant response occurred after injection with apoptin-expressing retroviral vector AdApt-VP3 in normal cells (Pietersen & Rutjes, unpublished data). Despite its nuclear translocation, apoptin was in most cases not toxic for the normal cells, as was also reported by others (Burek et al., 2005). In addition, in vivo kinase assays showed that this nuclear apoptin that was nontoxic to normal cells was not phosphorylated (Zhang, unpublished data), implying again that apoptin’s apoptotic activity is mediated not solely by nuclear compartmentalization but requires additional modifications (Danen-van Oorschot et al., 2003; Rohn & Noteborn, 2004). On the other hand, nuclear translocation and apoptosis induction in normal cells was not detected at all when apoptin protein was delivered as TAT-apoptin fusion (Guelen et al., 2004; Maddika et al., 2005), similar to the results of our protein microinjection experiments with MBP-apoptin (Fig. 1A). During DNA transfection, the nuclear membrane acts as a barrier to efficient DNA transfer, and DNA plasmid can thus be sequestered in the cytoplasm (Byrnes et al., 2002), which might result in DNA-activated cell stress signalling or induction of a viral defence pathway that facilitates apoptin’s subsequent nuclear targeting.
CHAPTER 5
108
Several versions of tag fusion for apoptin have been reported (Danen-van Oorschot et al., 2003; Guelen et al., 2004; Teodoro et al., 2004; Rohn et al., 2005). Plasmids encoding GFP-apoptin (Danen-Van Oorschot et al., 2003) and MBP-apoptin (Rohn et al., 2005) were mainly used by us in the early experiments. Surprisingly, fusion of GFP to full- length apoptin resulted in extensive nuclear accumulation of GFP-apoptin in human primary MSC (Rohn et al., 2005), as opposed to wild-type apoptin in parallel DNA microinjection experiments (Fig. 1B; Rohn et al., 2005), indicating that a tag may influence apoptin’s subcellular localization in normal cells. In addition, a C-terminally (but not an N-terminally) Strep-tagged apoptin fusion abolished apoptin’s phosphorylation in tumour cells, most likely due to structural mask on the phosphorylation site within apoptin (Voskamp & Noteborn, unpublished data). Therefore, fusion of a tag to apoptin protein for functional study needs to be designed and tested with caution.
Continuous culturing of normal cells invariably leads to phenotypic alteration of the cultures, possibly due to oxidative stress (Halliwell, 2003). For instance, the “Hayflick limit” in fibroblasts, i.e. replicative senescence after a certain number of cell divisions, may be largely an artefact of oxidative stress during cell culture (Halliwell & Whiteman, 2004). Therefore, we have always used normal human cells with a low passage number.
The most commonly used human normal cells in our apoptin studies were human primary fibroblasts, which were routinely used of passage lower than 15. In addition, we paid close attention to the appearance and behaviour of the cells. They should grow with a correct doubling time, and should look “clean and transparent and lack “lipid stress vesicle”
formation over the plasma membrane. It is also essential to test which medium is best for the cells prior to using them for experiments. For instance, our experience was that human NHDF fibroblasts preferred MEM medium to DMEM medium.
Of more than 10 types of normal human cells tested so far, we have not observed any cell- type-specific dependent toxicity of apoptin. Hence, we assume that apoptin’s nuclear translocation in normal cells largely results from the variations in experimental conditions, which we have not yet been able to pinpoint precisely.
REFERENCES
1. Burek M, Maddika S, Burek CJ, Daniel PT, Schulze-Osthoff K, Los M. Apoptin-induced cell death is modulated by Bcl-2 family members and is Apaf-1 dependent.
Oncogene, 2005, 25, 2213-2222.
2. Byrnes CK, Nass PH, Duncan MD, Harmon JW. A nuclear targeting peptide, M9,
improves transfection efficiency in fibroblasts. J. Surg. Res., 2002, 108, 85-90.
3. Danen-Van Oorschot AA, Fischer DF, Grimbergen JM, Klein B, Zhuang S, Falkenburg JH, Backendorf C, Quax PH, Van der Eb AJ, Noteborn MH. Apoptin induces apoptosis in human transformed and
EXPERIMENTAL CIRCUMSTANCES AFFECTING APOPTIN’S BEHAVIOUR
109 malignant cells but not in normal cells. Proc.
Natl. Acad. Sci. USA, 1997, 94, 5843-5847.
4. Danen-Van Oorschot AA, Zhang YH, Leliveld SR, Rohn JL, Seelen MC, Bolk MW, Van Zon A, Erkeland SJ, Abrahams JP, Mumberg D, Noteborn MH. Importance of nuclear localization of apoptin for tumor- specific induction of apoptosis. J. Biol.
Chem., 2003, 278, 27729-27736.
5. Danen-van Oorschot AA, Voskamp P, Seelen MC, van Miltenburg MH, Bolk MW, Tait SW, Boesen-de Cock JG, Rohn JL, Borst J, Noteborn MH. Human death effector domain-associated factor interacts with the viral apoptosis agonist Apoptin and exerts tumor-preferential cell killing. Cell Death Differ, 2004, 11, 564-573.
6. Guelen L, Paterson H, Gaken J, Meyers M, Farzaneh F, Tavassoli M. TAT-apoptin is efficiently delivered and induces apoptosis in cancer cells. Oncogene, 2004, 23, 1153-1165.
7. Halliwell B. Oxidative stress in cell culture:
an under-appreciated problem? FEBS Lett., 2003, 540: 3-6.
8. Halliwell B, Whiteman M. Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean? British Journal of Pharmacology, 2004, 142, 231-255.
9. Heilman DW, Green MR, Teodoro JG. The anaphase promoting complex: a critical target for viral proteins and anti-cancer drugs. Virus Genes, 2005, 31, 49-55.
10. Kooistra K, Peng D, Backendorf C, Noteborn MH, Zhang YH. Viral apoptosis inducer apoptin senses cell-type dependent nuclear trafficking of the transforming SV40 large T antigen., 2006, Submitted.
11. Leliveld SR, Zhang YH, Rohn JL, Noteborn MH, Abrahams JP. Apoptin induces tumor- specific apoptosis as a globular multimer. J.
Biol. Chem., 2003, 278, 9042-9051.
12. Maddika S, Booy EP, Johar D, Gibson SB, Ghavami S, Los M. 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., 2005, 118, 4485-4493.
13. Poon IK, Oro C, Dias MM, Zhang JP, Jans DA. A tumor cell-specific nuclear targeting
signal within chicken anemia virus VP3/apoptin. J. Virol., 2005a, 79, 1339-1341.
14. Poon IK, Oro C, Dias MM, Zhang J, Jans DA. Apoptin nuclear accumulation is modulated by a CRM1-recognized nuclear export signal that is active in normal but not in tumor cells. Cancer Res., 2005b, 65, 7059-7064.
15. Rohn JL, Zhang YH, Aalbers RI, Otto N, Den Hertog J, Henriquez NV, Van De Velde CJ, Kuppen PJ, Mumberg D, Donner P, Noteborn MH. A tumor-specific kinase activity regulates the viral death protein Apoptin. J. Biol. Chem., 2002, 277, 50820-50827.
16. Rohn JL, Noteborn MH. The viral death effector Apoptin reveals tumor-specific processes. Apoptosis, 2004, 9, 315-322.
17. Rohn JL, Zhang YH, Leliveld SR, Danen- van Oorschot AA, Henriquez NV, Abrahams JP, Noteborn MH. Relevance of apoptin's integrity for its functional behavior. J. Virol., 2005, 79: 1337-1338.
18. Tavassoli M, Guelen L, Luxon BA, Gaken J.
Apoptin: Specific killer of tumor cells?
Apoptosis, 2005, 10, 717-724.
19. Teodoro JG, Heilman DW, Parker AE, Green MR. The viral protein Apoptin associates with the anaphase-promoting complex to induce G2/M arrest and apoptosis in the absence of p53. Genes Dev., 2004, 18, 1952-1957.
20. Wadia JS, Wagner MV, Ezhevsky SA, Dowdy SF. Apoptin/VP3 contains a concentration-dependent nuclear localization signal (NLS), not a tumorigenic selective NLS. J. Virol., 2004, 78, 6077-6078.
21. Zhang YH, Kooistra K, Pietersen A, Rohn JL, Noteborn MH. Activation of the tumor- specific death effector apoptin and its kinase by an N-terminal determinant of simian virus 40 large T antigen. J. Virol., 2004, 78, 9965-9976.
22. Zhang YH, Leliveld SR, Kooistra K, Molenaar C, Rohn JL, Tanke HJ, Abrahams JP, Noteborn MH. Recombinant Apoptin multimers kill tumor cells but are nontoxic and epitope-shielded in a normal-cell- specific fashion. Exp. Cell Res., 2003, 289, 36-46.
23. Zhuang SM, Shvarts A, van Ormondt H, Jochemsen AG, van der Eb AJ, Noteborn MH. Apoptin, a protein derived from chicken
CHAPTER 5
110
anaemia virus, induces p53-independent apoptosis in human osteosarcoma cells.
Cancer Res., 1995, 55: 486-489.
