Overall, we found that a primate virus can produce diverse abnormal proliferating cells by cell fusion and that proliferation of these cells is favored by expression of oncogenes or by a mutated tumor suppressor, p53. Whether proliferating hybrids are produced by viruses in vivo, and whether these hybrids can evolve into cancerous cells, remains to be determined.
At least two sets of observations suggest that viruses produce not only moribund syncytia but also proliferating hybrids. For example, human cancer cells grafted into rodents produce tumors that partially or completely consist of host-human hybrids and host tumor cells that appear to be derived by spontaneous fusion, which is a term used to describe fusion with an unknown cause (Goldenberg et al., 1974
; Pathak et al., 1997
; Mortensen et al., 2004
). Interestingly, mouse cancer cell lines passaged in mice can increase or acquire their metastatic potential by spontaneous fusion to host cells (for review see Duelli and Lazebnik, 2003
Why these tumor cells are fusogenic is unclear. Our observations suggest an explanation: fusion is caused by viruses or viruslike particles. Indeed, not only are retroviruses ubiquitous in mice, but some viruses were specifically associated with xenografts and the resulting tumors (Bowen et al., 1983
; Ristevski et al., 1999
). We found that 7 out of the 20 human tumor cell lines that we tested released a particulate fusogen, which is consistent with the possibility that these tumor cell lines released fusogenic viruses (unpublished data).
Another example of cell fusion that remains unexplained is the production of hybrids between bone marrow stem cells and differentiated somatic cells (O'Malley and Scott, 2004
; for review see Vignery, 2005
). Bone marrow stem cells can give rise not only to somatic differentiated cells but also to gastric cancer by a mechanism that is unclear (Houghton et al., 2004
). One possibility that was raised (Marx, 2004
) but not tested is that oncogenic transformation in this case was also caused by fusion. Again, viruses may be the fusogen, a possibility that would also explain the puzzlingly low frequency of stem-cell hybrids (10−5
; O'Malley and Scott, 2004
An intriguing question is why hybrids of stem cells are capable of proliferating. Our observations suggest that the answer may be in the plastic cell cycle regulation in stem cells, which makes them similar to embryonic or to partially transformed cells (Attar and Scadden, 2004
). Therefore, the ability to survive and proliferate after fusion, rather than to fuse, may be a particular characteristic of stem cells that underlies fusion-mediated transdifferentiation, and perhaps cancer, and allows one to detect these hybrids in vivo. Given that fusing somatic cells is being considered as a therapeutic approach (Cowan et al., 2005
), it is of practical interest to know for certain that this fusion does not produce malignant progeny.
Whether hybrids observed in animal models occur in humans is unclear, although the reports of premature chromosome condensation (Kovacs, 1985
) and hybrids between transplanted and host cells (Chakraborty et al., 2004
) are consistent with this possibility. There is little doubt, however, that virus-induced cell fusion in people is common, as syncytia are a feature of many viral infections, from the respiratory syncytial virus to human immunodeficiency virus.
What kind of viruses can produce proliferating hybrids?
Our finding that a virus can produce proliferating hybrids was fortuitous. MPMV is neither cytotoxic nor cytostatic in human cells and in our system primarily produced dikaryons, which are more likely to result in proliferating hybrids than cells with more nuclei. It is unlikely that these characteristics are unique for this virus.
One group are the viruses that, like MPMV, are considered harmless and therefore are studied much less intensely than obvious pathogens. For example, human T cell lymphotropic virus type 1 (HTLV-1) is harmless to most of its carriers, and its association with cancer was uncovered only by epidemiological studies. However, cytotoxic viruses could also produce hybrids by fusion with noninfectious viral particles, which are often more abundant than infectious particles, or if infection is restricted by host factors. In either case, the hybrids may have no traces of the virus, implying that the viral etiology of virus-induced hybrids could be difficult to determine. Because oncogenes can allow proliferation of fused cells, fusogenic viruses carrying such oncogenes (e.g., myc) may not only fuse cells but also allow the hybrids to proliferate. Retroviruses may achieve a similar effect by insertional mutagenesis.
The ability to form dikaryons, which are more likely to have proliferating progeny, may depend not only on the virus but also on how and from what kind of cell it is released. For example, if a virus is released as a part of exosomes, it may carry cellular proteins, such as tetraspanin CD9 and -81, that facilitate fusion of cells. Tetraspanins may also be acquired by the viruses that are released from tetraspanin microdomains of the plasma membrane (Hemler, 2003
; Martin et al., 2005
Endogenous retroviruses (ERVs), whose sequences comprise at least 8% of the human genome (Griffiths, 2001
), are also candidates as pathogenic fusogens. At least three Env proteins of human ERV are fusogenic, two of which, Syncytin 1 and 2, are normally expressed only in the placenta, where they mediate fusion required for formation of the syncytiotrophoblast. The third protein, Env(P)b, whose physiological function is unknown, is expressed ubiquitously (Blaise et al., 2005
). Interestingly, expression of ERVs is regulated by a variety of factors, including DNA damage, mitogens, and hormones (Taruscio and Mantovani, 2004
). Overall, the possibility that viruses produce proliferating hybrids should at least be considered in developing cancer treatments that use fusogenic viruses to kill cells or as vectors for gene therapy.
Are exosomes misidentified viruses?
Because exosomes and some viruses are so similar, at least some particles reported as exosomes may be misidentified viruses. This possibility has practical implications because exosomes are used in cancer therapy (for review see Chaput et al., 2004
). Indeed, early reported compositions of exosomes did include retroviral proteins (Thery et al., 1999
). As we found, identifying vesicles as exosomes or viruses by mass spectrometry depends on the protocol used. Defining parameters such as size, shape, and density in sucrose gradients are also conspicuously similar (for review see Fine and Schochetman, 1978
; Thery et al., 1999
). The morphology of MPMV (Smith et al., 1978
), a pleomorphic virus, is difficult to distinguish from that of exosomes.
Cell fusion as a link between viruses and carcinogenesis
A current view is that retroviruses transform cells either by integrating into the cellular genome, thereby affecting normal gene expression or modifying cellular genes, or by introducing oncogenes in the genome of infected cells. However, the transformation induced by viruses is unlikely to be limited by these mechanisms, as indicated by the recent findings that the Env proteins of Jaagsiekte virus and enzootic nasal tumor viruses are causative agents of infectious cancer in animals (Wootton et al., 2005
Whether retroviruses cause human cancer is a subject of discussion. The claim that they do is based on epidemiological data (for review see Mant et al., 2004
), whereas the claim that they do not is based on the argument that viruses fail the requirements imposed by Koch's postulates (Duesberg, 1987
; Blaho and Aaronson, 1999
; Talbot and Crawford, 2004
). These postulates, as applied to cancer, argue that a candidate virus must be present in cancer but not in healthy cells, must be isolated from the cancer cells, must cause oncogenic transformation if introduced into normal cells, and must be present in these cells once they are transformed. Indeed, if these standards apply, the proof that viruses are etiological agents of human cancer falls short (for review see Mant et al., 2004
). For example, only 1% of the people infected with HTLV-1 develop cancer with no apparent correlation between carcinogenesis and the virus integration sites (Hanai et al., 2004
), whereas another small fraction of the carriers develop a disease unrelated to cancer.
However, Koch's postulates are valid only if the causal relationship between viruses and oncogenic transformation is as direct as that between viruses and infectious diseases, which is an assumption that is consistent with the current view of viral oncogenesis. If the causal relationship between viruses and oncogenic transformation includes events with random outcomes in respect to carcinogenesis, such as cell fusion and abnormal mitoses, and the virus may even be absent in the cells it produced, then the cause–effect relationship between viruses and cancer is intrinsically stochastic (), which would mean that Koch's postulates do not apply. Therefore, the observed correlations between viral infections and human cancer may be more than coincidental, even though because of its random nature it may be impossible, in principle, to establish a mechanistic link by analyzing only cancer cells.
Figure 9. Cell fusion as a link between viruses and carcinogenesis. Potential implications of our findings to carcinogenesis could be summarized by the following speculative model. Although illicit cell fusion induced by viruses may be a frequent and common event, (more ...)
Irrespective to the origin of cancer cells, cell fusion has the potential to promote diversity of transformed cells, as our results and previous studies have demonstrated. In this case, the frequency with which proliferating hybrids are produced would be even higher because at least one of the fusion partners has a deregulated cell cycle and, perhaps, greater survival capabilities.
Overall, the evidence presented in this study suggests a mechanistic link between viral infections and the genetic and epigenetic instability model of carcinogenesis. Whether this link contributes to carcinogenesis or cancer progression remains to be determined.