The human diploid fibroblast has long been popular in studies of neoplastic transformation because of its ease of isolation from human subjects and its central role in understanding replicative senescence and crisis [25,26
]. Attempts to convert normal human fibroblasts into cancer cells by direct genetic modification failed until Hahn et al. [1
] reported that four genes—SV40 LT, SV40 ST, Ras
, and hTERT
—were required for this transformation. In particular, in that study and in previous reports [15
], the combination of SV40 LT and Ras was nontumorigenic by the conventional criterion of formation of tumors following subcutaneous injection of the cells in immunodeficient mice. Further experiments in the same model—human fibroblasts and SV40 LT/SV40 ST/Ras/hTERT—have confirmed and amplified the conclusion that these is a minimal set of genes required for neoplastic transformation [27–33
However, we demonstrate here that this conclusion is not valid when early-passage primary human fibroblasts are transduced with SV40 LT and Ras and implanted beneath the kidney capsule rather than under the skin of immunodeficient mice. Early-passage human fibroblasts transduced with SV40 LT and RasG12V produced tumors that were invasive and sometimes metastatic. Thus, using experimental conditions that are as close to the in vivo situation as possible—the use of early-passage cells and formation of the tumor within an internal organ—both hTERT and SV40 ST are eliminated from the minimal set of genes required to convert human fibroblasts into cancer cells.
The present study used nine different strains of primary human fibroblasts—all from normal donors and all used at the earliest PD available. In agreement with previous data on early-passage fibroblasts [21
], we found that Ras had a positive effect on growth; in particular, we found that Ras-transduced cells exhibited anchorage-independent growth. Later-passage cells show a radically different response to Ras—in those cells, Ras causes senescence [34
]. This has been puzzling, given the central role of oncogenic Ras in increasing proliferation, invasion, angiogenesis, and other features of tumors in vivo [2,3
]. Ras-induced senescence requires the activity of p16INK4A
]. The passage-dependent induction of senescence by RasG12V
is correlated with an increase in p16 as a function of PDL [21,31
]. This increase in p16 appears to be a response to oxidative or other forms of damage incurred during growth in culture [39
]. These results question whether p16-dependent Ras-induced senescence represents a barrier that must be overcome for cells to respond to the protumorigenic effects of Ras in a living tissue. It is important to define sets of genes that are required for neoplastic transformation using early-passage cells, thereby avoiding artificial barriers to transformation that have been acquired by the cells during growth in culture.
Evidently, the microenvironment in the kidney is ideal for the survival, growth, and vascularization of both normal and neoplastic cells. The critical features of this microenvironment have not been definitively identified; important features may be the proximity to abundant capillaries within the kidney and the positive interstitial fluid pressure [40
]. This site is also ideal for revealing invasive behavior of tumorigenic cells, as malignant cells grow between renal tubules, eventually destroying the entire organ. We confirmed earlier observations that normal human fibroblasts, as well as fibroblasts expressing only SV40 LT or only Ras, have no malignant properties when transplanted in the kidney. They form a thin layer on top of the kidney parenchyma. A clear boundary with the kidney is maintained and there is no sign of invasion (data not shown). A relevant question is whether the kidney is an “ectopic” site for the growth of neoplastic fibroblasts and that the subcutaneous site might be more “physiological.” However, it should be noted that fibroblasts occur in all organs of the body and could potentially (although rarely) form a tumor in any organ. Conversely, subcutaneous injection actually places the cells beneath the panniculus carnosus and not within the dermis; they are therefore not actually within the skin.
The present experiments lead to the conclusion that both hTERT and SV40 ST are dispensable for tumorigenic growth of human fibroblasts. However, both genes are required for tumorigenic growth of cells beneath the skin, and hTERT is also required for long-term tumorigenicity of SV40 LT/Ras-transduced cells. The latter effect of hTERT is specific for the rescue of cells that have reached crisis. Whereas SV40 LT/Ras-transduced cells are clearly malignant, as evidenced by invasive and metastatic growth, which may result in the death of the animal, they are nevertheless not immortal. Growth of the tumors eventually ceases because of telomere shortening, which leads to crisis. Crisis was demonstrated by nuclear DNA damage foci and mitotic catastrophe, and by more specific indications of telomere dysfunction such as anaphase bridges and internuclear chromatin strings. Cells were rescued from this state by expression of hTERT, which conferred telomerase activity and caused telomere lengthening. Under these circumstances, hTERT restores the malignant potential of the cells and permits serial transplantation of the tumor through multiple host animals. The incidence of DNA damage foci in tumors was also greatly reduced (data not shown).
Thus, immortalization, as a feature of cancer cells, should be distinguished from other features of malignant cells. In order for cells to form a lethal cancer, they must have acquired the ability to bypass normal controls on cell proliferation, the ability to invade and destroy organs, and the ability to form distant metastases. Although other properties of cancer cells are commonly observed, such as resistance to apoptosis and immortality, there is no a priori
reason for those properties to be essential for cells to form a lethal cancer. In the SV40 LT/SV40 ST/Ras/hTERT model of neoplastic transformation of human fibroblasts, the absolute requirement for hTERT is problematic. If immortalization is the specific property conferred by hTERT, then one would expect that cells with relatively long telomeres should still be converted to tumor cells by SV40 LT/SV40 ST/Ras and that such cells would develop a requirement for telomerase only when telomeres have shortened to a critical extent, as shown here in the subrenal capsule assay. In contrast, in the subcutaneous assay, the SV40 LT/SV40 ST/Ras combination produced no tumors in extensive trials [1,29,41–43
These data suggest that, in the subcutaneous site, hTERT has an essential role in tumorigenesis by conferring properties on cells, other than immortalization. The requirement for hTERT may reflect one or more of the many other reported effects of high-level hTERT expression in cells, including stimulation of cell proliferation and antiapoptotic effects [14
]. Further studies are needed to define the cellular and molecular processes/pathways that must be targeted to permit the survival and growth of cells in the subcutaneous site, which are not needed in the subrenal capsule site. They could include resistance to stresses, such as osmotic stress, lack of nutrients, oxidative stress, and hypoxia. Because these genetic modifications are not required for tumorigenicity per se
, genes that have been identified in the past as oncogenes (or classified as nononcogenic) based on subcutaneous injection experiments need to be restudied using growth of cells within an internal organ.
In summary, the minimal set of genetic changes required for tumorigenicity of human fibroblasts was assessed by implanting cells in immunodeficient host animals at a site that permits optimal cell survival, using early-passage cells that have not acquired artefactual barriers to neoplastic transformation. The cellular pathways targeted by SV40 large T antigen (p53 and pRb) and those targeted by activated Ras represent a minimal set of genetic alterations required for the conversion of normal human fibroblasts into cancer cells.