The present observations indicate that the actions of hTERT and oncogenic H-Ras, together with three SV40-encoded functions, are sufficient for the transformation of human fibroblasts and HEK cells. The operational definition of transformation used here implies the ability of cells to form tumors in immunocompromised mice without showing the additional phenotypes of invasiveness or metastatic ability. At least two of these necessary viral functions—those affecting pRB and p53—can be replaced by mutant alleles of cellular genes. Indeed, our success in replacing LT with a combination of mutant cellular genes that selectively disrupt pRB and p53 function indicates that all of the other functions exerted by LT are not required for the induction of tumorigenic growth of human cells.
Previous studies have identified and characterized the elements of the SV40 ER required for the immortalization and transformation of various types of rodent cells. While the experimental transformations of human, mouse, and rat fibroblasts with SV40 share many fundamental features, several differences in the contribution of specific elements of the SV40 ER to human and rodent cell immortalization and transformation are apparent. Although some of these discrepancies likely represent differences in the experimental protocols used by various research groups, it is clear that rodent cells are much more susceptible to transformation than are human cells (42
). Identifying and understanding these species-specific differences in the functioning of the various SV40 ER oncoproteins will help elucidate the molecular mechanisms that lead to immortalization and transformation during the formation of human cancers.
In particular, the frequency of spontaneous immortalization upon extended passage in vitro differs considerably between rodent and human cells. Rodent cells are readily immortalized, in contrast to most types of human cells, which virtually never undergo spontaneous immortalization in culture (87
). Paralleling this difference in the rate of spontaneous immortalization, several studies have demonstrated that expression of the bipartite p53-binding domain of LT alone is sufficient to extend the life span of most primary MEF (79
). These findings are consistent with observations that implicate the p19ARF-MDM2-p53 pathway as a critical determinant of the murine cell life span (24
). In contrast, our observations obtained by using human fibroblasts and HEK cells (Fig. ) confirm a previous report (71
) that the binding and sequestration of both pRB and p53 by LT are necessary to allow human cells to bypass replicative senescence in the absence of telomerase expression. Even though most murine cells express constitutive telomerase activity (59
) and maintain extremely long telomeres (35
), this difference in telomere biology between human and rodent cells fails to explain the additional requirement for the inactivation of the pRB pathway for human cell immortalization. Although Conzen and Cole have reported that expression of LT mutant forms containing internal deletions of the pRB-binding site or of the J domain immortalized primary MEF with poor efficiency (8
), we and others (71
) have never obtained immortalized human cells expressing the LT K1 mutant form that is unable to bind pRB. In consonance with prior studies using MEF (8
), we found that ST does not play a significant role in the immortalization of human cells.
The transformation of both primary human and murine cells by SV40 LT requires the inactivation of both the pRB and p53 pathways (21
). In addition, prior studies have suggested that the J domain of LT plays an important role in the transformation of MEF (76
). Expression of LT J domain mutant forms in MEF abrogates the ability of such cells to grow to a high density or to grow under limiting serum conditions compared to cells expressing wild-type LT yet did not affect the ability of such cells to grow in an AI manner in soft agar (79
). Since the J domain was dispensable for these phenotypes in MEF doubly deficient for the pRB-related proteins p107 and p130, the J domain appears to alter the ability of LT to inactivate these pRB-related proteins (79
). Moreover, the J domain of ST is functional since expression of ST can complement LT J domain mutant forms to transform MEF (45
). However, in the experimental model of human cell transformation presented here, by expressing a mutant ST lacking a functional J domain (ST H42Q or ST F88-174) in combination with cyclin D1, the R24C CDK4 mutant form, p53DD, hTERT
, and ras
genes, we were able to transform primary fibroblasts to a tumorigenic state without introduction of a J domain (Fig. ), indicating that an intact LT J domain is not required for immortalization, growth in soft agar, or the formation of tumors in human cells. Although the reason for this discrepancy between human and murine cell transformations remains obscure, it is possible that the relative contribution of each of the pRB-related proteins to suppression of transformation differs between MEF and human fibroblasts or that the particular combination of transforming elements used here permits transformation without the requirement for a functional J domain.
These observations raise the question of whether perturbation of the pathways examined here (involving the pRB, p53, PP2A, hTERT, and Ras proteins) is also sufficient for the transformation of these and other types of human cells. We have accumulated three lines of evidence that persuade us that the experimental disruption of these pathways does, indeed, suffice to convert normal human cells to tumorigenicity. First, we introduced these genes by retroviral vectors, ensuring that polyclonal populations were produced (21
); this substantially reduced the possibility that rare, secondary transforming mutations occurring in still unknown genes are required to act, in concert with the introduced genes, to achieve transformation. Importantly, the populations of tumor cells that emerge from these manipulations and, indeed, from tumor-bearing animals retain evidence of polyclonality (13
). Second, when transformed cells that have formed tumors in a host are subsequently reimplanted in a second host animal, these cells form tumors with kinetics that are identical to those of cells that have never been implanted in a host mouse (13
). Such observations make it highly unlikely that the tumorigenic phenotype of these cells depends on the acquisition of an additional, mutant allele during their passage in vivo. Finally, we have demonstrated that many of the transformed HEK cells lack evidence of widespread genomic instability, which might yield a plethora of secondary mutations that would confound the genetic characterization of the tumorigenic cells studied here. Instead, these transformed HEK cells were largely diploid, with no evidence of microsatellite instability or translocations (95
). Taken together, these various observations provide persuasive evidence that these five alterations are sufficient to achieve the tumorigenic transformation observed by us (13
) and others (60
The identification of ST as an essential participant in the transformation assay has permitted us to begin to enumerate the major pathways that contribute to human cell transformation and explains the previously observed inability of the HPV-16 E6 and E7 oncoproteins to transform human fibroblasts when expressed in combination with hTERT and oncogenic H-Ras (46
; Fig. and ). ST has long been known to play a role in transformation by SV40. SV40 mutants that are incapable of producing ST show a diminished ability to transform rodent cells (61
). In addition, De Ronde et al. (10
) showed that similar SV40 mutants were unable to transform human fibroblasts when focus-forming ability was gauged but could transform murine fibroblasts, albeit less efficiently than wild-type SV40. This dependence on ST was more apparent in assays performed when cells were at a high density (93
) or when LT was expressed at lower levels (2
). More recently, others have demonstrated that human mesothelial cells expressing both SV40 LT and ST are more easily transformed by subsequent exposure to carcinogens than are cells expressing only LT (4
). Indeed, transgenic mice expressing both LT and ST under the control of the mouse mammary tumor virus promoter developed a broader spectrum of epithelial cancers than did mice expressing only LT (7
). While these studies investigated the role of ST in the context of LT expression, the data presented here demonstrate that the expression of ST is also required to complement the disruption of the pRB and p53 pathways even if these pathways are disrupted through other genetic means. Taken together, these observations demonstrate the important contribution of ST, ostensibly through its perturbation of cellular PP2A, to the transformation of human cells.
The present observations raise the question of whether alterations of PP2A function also occur during the pathogenesis of spontaneously arising human tumors. Recently, two groups have identified a subset of lung cancers, breast cancers, and malignant melanoma that show loss of heterozygosity at chromosome 11q22-24, which contains the genetic locus specifying the PP2A β56 A subunit (6
). In approximately 15% of such tumors, further mutations or deletions affecting conserved amino acids in the surviving β56 allele were identified that would be predicted to perturb PP2A function by disrupting the interaction of this PP2A subunit with the other two subunits of the PP2A holoenzyme. These observations suggest that alterations in PP2A can play an important role in the development of spontaneously arising human cancers. However, these tumors represent only a small proportion of human cancers and it remains to be determined whether systematic efforts to survey the array of PP2A subunit genes in human tumor genomes will reveal frequent disruptions of the normal signaling regulated by this enzyme complex.
Expression of ST induces increased cell proliferation in cells immortalized with LT and hTERT
(Fig. ). In addition, using cells expressing or lacking constitutive H-Ras signaling, we showed that ST induces cell proliferation independently of the activity of the H-Ras signaling pathway. While cells that express only LT, hTERT, and Ras are immortal, these cells double three times more slowly in vitro than do corresponding cells that also express ST, and these ST-negative cells do not form tumors, even after 6 months of observation postimplantation. Consistent with these observations, transient expression of ST in CV-1 monkey cells also stimulated cell growth, correlated with an increase in the phosphorylation of the ERK (extracellular signal-regulated protein kinase) and MEK (mitogen-activated protein/ERK kinase) kinases in vitro (75
The stimulation of cell proliferation induced by the presence of ST cannot explain the full contribution of ST to cell transformation. In particular, we observed the growth-stimulatory effects of ST when cells were grown in the absence of anchorage to a solid substrate (Fig. ) or when they were grown under conditions of amino acid deprivation (Fig. ). Comparable conditions are likely to exist when cells are placed experimentally into host animals to assess tumorigenicity and may also mimic conditions that nascent tumor cells face early in the formation of a spontaneously arising tumor. Taken together, these observations suggest that ST disrupts one or more cellular regulatory checkpoints that limit cell proliferation when cells encounter suboptimal growth conditions.
ST mutant forms previously shown to disrupt PP2A binding also fail to cooperate to transform human cells (Fig. ). While it remains formally possible that a second host cell protein shares the same binding site on ST, these observations suggest that ST must interact with PP2A in order to collaborate in the transformation process. However, the precise biochemical alterations that result from this interaction remain elusive. Genetic deletion of PP2A in eukaryotic cells results in cell death (14
); thus, complete inhibition of PP2A is not possible. Furthermore, since PP2A represents a large and complex family of enzymes, the resulting holoenzymes created through the combinatorial associations of its various alternative subunits may be responsible for the dephosphorylation and regulation of hundreds of distinct cellular phosphoprotein substrates. For these reasons, it is clear that further investigation is required to identify the substrate proteins whose dephosphorylation by PP2A must be inhibited by ST in order for cell transformation to occur.
We speculate that perturbation of the five pathways described here will be found in most, if not all, types of human tumors. If so, many of the genetic lesions found in human tumor cell genomes may one day be rationalized in terms of their effects on these five pathways. Clearly, these pathways do not elicit two changes that are frequently associated with human tumor cells: those that destabilize the genome and thereby accelerate the rate of tumor pathogenesis and those that confer the phenotypes of invasiveness and metastasis. Furthermore, since successful transformation required H-Ras expression at levels 10- to 20-fold higher than those seen in human tumor samples (13
), the overexpressed Ras oncoprotein may function in a manner that is qualitatively similar to the actions of physiologic Ras levels, merely permitting the rapid growth of tumors in the 3- to 6-week time span of these experiments. Alternatively, the high levels of H-Ras expression used here may act in a fashion that is qualitatively different from that of the Ras oncoprotein in human tumors; by virtue of its higher levels of expression, it may activate additional downstream signaling pathways whose activation contributes to the observed tumorigenic growth.
The increased availability of cultures of primary human epithelial, mesenchymal, neuroectodermal, and hematopoietic cells from various tissues will facilitate the construction of a library of human tumor cells by genetic strategies similar to that used here. Further replacement of the genetic elements used in the present studies with mutant genes found in particular types of human cancers will create models that will increasingly mimic the phenotypes of naturally arising human tumor cells and will permit investigation of the additional changes required to allow cells to invade and metastasize. Finally, the availability of genetically defined human tumor cells will facilitate the testing and validation of therapies in which cancer cell responses to specific therapies will be rationalized in terms of the genetic and biochemical lesions introduced into such cells.