Many human cancer cells lack telomerase activity but nevertheless maintain telomeres via a process termed “alternative lengthening of telomeres” (ALT). Despite being immortal and having a telomere maintenance mechanism, ALT+ human fibroblasts require telomerase reverse transcriptase (hTERT) for tumor formation in immunodeficient mice when tested by s.c. injection. Here we show that three ALT+ human SV40-immortalized fibroblast cell lines require only oncogenic RasV12G to be converted to a fully tumorigenic state. When cells were implanted beneath the kidney capsule of immunodeficient mice, they invaded the kidney and neighboring organs and metastasized to the lungs. RasV12G-expressing ALT+ cells remained completely telomerase negative. Introduction of hTERT conferred strong telomerase activity but did not appreciably change the malignant properties of the cells. However, when cells were tested by s.c. injection, RasV12G-transduced ALT+ cells did not form tumors, and in this site, hTERT was required for tumorigenicity. These data show that when the s.c. injection method is used as an assay for tumorigenicity, hTERT may be artifactually scored as an oncogene; the subrenal capsule assay shows that ALT, as a telomere maintenance mechanism, is equivalent to hTERT in neoplastic transformation of human cells by oncogenes.
Immortalization of human cells requires a telomere maintenance mechanism. In the germ line, in stem cells, and in most cancer cells, telomere length is maintained by the telomerase ribonucleoprotein complex (1). Some telomerase-negative cancer cells maintain their telomeres by a recombination-based process termed “alternative lengthening of telomeres” (ALT; refs. 2, 3). Because both telomerase and ALT permit immortalization, it was generally assumed that these processes would be equivalent in oncogene cooperation models of human tumorigenesis. Primary human fibroblasts can be transformed to a fully malignant state by the combination of SV40 large T (LT) antigen, small t (ST) antigen, oncogenic Ras, and human telomerase reverse transcriptase (hTERT; refs. 4, 5). If the function of hTERT in this combination is to confer a telomere maintenance mechanism then it would be expected that ALT+ cells would not require hTERT for neoplastic transformation. However, SV40-immortalized ALT+ cells, which express LT and ST, were found to require both oncogenic Ras and hTERT to become tumorigenic (6). Based on these findings, it was concluded that hTERT does not contribute to tumorigenicity solely by maintaining telomeres but rather confers other properties on cells that are required for neoplastic transformation. hTERT can exert a variety of effects in cells other than telomere maintenance (7). However, in these experiments and in most experiments that have involved the conversion of human cells to cancer cells by defined genetic manipulations, tumorigenicity was assayed as tumor development when cells were injected under the skin of immunodeficient mice. We have shown that using this site of the body for tumorigenesis assays may produce misleading results; a larger number of genetic changes are needed for growth of cells into a tumor under the skin than in the subrenal capsule assay (8). Based on these results, we hypothesized that SV40-immortalized ALT+ cells would require only oncogenic Ras for complete transformation to a tumorigenic state.
Materials and Methods
Growth of cells and retroviral transduction. Three SV40-immortalized ALT+ human fibroblast cell lines were used: GM847 (LN-SV; ref. 9), GM639 (10), and WI-38 VA13 subline 2RA (11). Control normal human foreskin fibroblasts (HCA2) were obtained from Dr. Olivia Pereira-Smith (University of Texas Health Science Center at San Antonio). The RasV12G-encoding retrovirus is of the LX type (12) and also expresses GFP from an internal promoter (8). The Phoenix cell line (amphotropic) was used for production of retroviral particles (8, 13). Pure populations of transduced cells were prepared by flow cytometry. More than 98% of the cells were fluorescent after sorting. Sorted cells were expanded in culture for transplantation or for biochemical studies. No drug selection was used to introduce Ras, but when pBabe-hTERT was used, cells were selected with 1 μg/mL puromycin. Western blotting and telomerase repeat amplification protocol (TRAP) assays were done on the transduced cells as previously described (8).
Transplantation of cells in RAG2−/−,γc−/− mice. RAG2−/−,γc−/− mice at an age of >6 weeks (∼25 g body weight) were used in these experiments. Procedures were approved by the institutional animal care committee and were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals. We transplanted 2 × 106 cells beneath the kidney capsule as previously described for adrenocortical cells (14–16). S.c. cell implantation was done using a 29-gauge needle. Potential leakage of cells from the injection site was monitored by illumination with a 470-nm light source (Lightools Research, Encinitas, CA).
Animals were sacrificed at various times following transplantation, as described in Results. Tumors were visualized by fluorescence using a 470-nm light source in conjunction with a low level of white light. This combination allowed the direct visualization of the fluorescent tumor cells together with the nonfluorescent organs (8).
GM847 cells, an immortalized ALT+ cell line derived from SV40-infected human fibroblasts, were transduced with a retrovirus encoding RasV12G and GFP. Transduced cells were selected by fluorescence-activated cell sorting. The potential tumorigenicity of the transduced cells was then tested by injection into the subrenal capsule space of immunodeficient mice. GM847 cells are immortal but lack telomerase activity (2). In view of a previous report that despite being immortal, GM847 cells required expression of both RasV12G and hTERT for tumorigenicity in immunodeficient mice (6), we compared the properties of RasV12G-transduced cells and RasV12G/hTERT-transduced GM847 cells. Both Ras-transduced and Ras/hTERT-transduced GM847 cells formed rapidly growing tumors when implanted in the kidney (Fig. 1A-B). Lung metastases were also observed in both groups of animals. The retrovirally transduced cells expressed high levels of Ras, as well as SV40 LT and ST, as expected based on previous studies with this retrovirus (ref. 8; Fig. 1C). Transduction of cells with RasV12G did not confer detectable telomerase activity (Fig. 1D). As expected (8), cells transduced with hTERT as well as Ras were strongly telomerase positive. We isolated GM847 cells from a subrenal capsule tumor by enzymatic dissociation and flow sorting. Like pretransplantation cells, these cells did not have detectable telomerase activity (Fig. 1D).
We evaluated potential differences in invasive behavior between RasV12G-transduced and RasV12G/hTERT-transduced cells in the subrenal capsule assay. Both cell populations were highly invasive; qualitatively, no differences were seen between the two. Both cell types showed invasion into the kidney, pancreas, muscle, fat, and occasionally liver (Fig. 2).
As shown in Fig. 1, expression of hTERT did not appreciably change the tumorigenic properties of RasV12G-transduced GM847 cells when tested in the subrenal capsule assay. However, based on previously published data, we anticipated that GM847 cells would require hTERT as well as Ras to form tumors when cells were injected s.c. rather than under the kidney capsule (6). We confirmed that Ras-transduced GM847 cells did not form tumors when injected s.c. Small fluorescent blebs under the skin were seen after injection of the cells, which disappeared over the next 2 weeks. However, GM847 cells transduced with Ras and hTERT formed invasive, continuously growing tumors in this site (Fig. 2).
The requirement for hTERT in s.c. tumor growth shown in this experiment might indicate that tumors cannot grow s.c. without hTERT, or might indicate a need for hTERT only when cells are injected as a cell suspension. To test this, RasV12G-transduced GM847 tumors were grown in the kidney. Small (1 mm) fragments were excised from such tumors and were implanted under the skin. These fragments enlarged to 1-cm diameter and invaded surrounding tissues (Fig. 2). However, when hTERT-negative tumors grown in the kidney were dissociated into a single-cell suspensions and injected under the skin they were unable to re-form tumors.
The results obtained on RasV12G-transduced GM847 cells indicated that the combination of SV40 LT, SV40 ST, and Ras is sufficient to confer malignant properties in an ALT+ cell line and that hTERT is not required, except to initiate tumors when cells are injected s.c. We hypothesized that this conclusion should be valid for ALT+ cell lines generally. We investigated whether two other SV40-immortalized ALT+ cell lines could form tumors without hTERT in the subrenal capsule assay (Table 1). Ras-transduced GM639 and VA13 cells both were tumorigenic in this site. However, none of the Ras-transduced cell populations were able to form tumors when injected s.c.
|Fibroblast strain .||Genetic modification .||Tumor incidence|
|.||.||S.c. .||Subrenal capsule .|
|GM847||Ras + hTERT||5/7||7/7|
|Fibroblast strain .||Genetic modification .||Tumor incidence|
|.||.||S.c. .||Subrenal capsule .|
|GM847||Ras + hTERT||5/7||7/7|
NOTE: Three SV40-immortalized ALT+ cell lines were transduced with RasV12G; the incidence of tumors following s.c. injection was compared with that in the subrenal capsule assay. The formation of tumors by Ras/hTERT-transduced GM847 cells is also shown. Mice were sacrificed at 35 days following cell transplantation.
We show here that ALT+ cell lines derived from SV40-infected human fibroblasts do not require hTERT to become tumorigenic when transduced with RasV12G, although hTERT is needed for tumor growth when cells are implanted under the skin of immunodeficient mice. The requirement for hTERT for s.c. tumor growth cannot be explained by hTERT conferring a telomere maintenance mechanism on the cells, because evidently ALT+ cells do not lack such a mechanism; they are able to maintain their average telomere length indefinitely in culture without crisis or senescence (2, 3). The fact that hTERT is needed for growth of ALT+ cells only when they are injected under the skin and not when they are inserted into an internal organ indicates that hTERT acts by enhancing cell survival or other functions needed for s.c. growth. Various effects of ectopic hTERT expression have been noted (7). For example, in ALT+ cells, expression of hTERT increased cell proliferation under conditions of cellular stress (0.1% serum, 2.5 mmol/L glucose, and 0.4% oxygen; ref. 6). The most likely influence of hTERT is on the initial survival of the cells when they are injected under the skin, because we show here that hTERT-negative tumors are able to grow s.c. when serially transplanted from a tumor growing in the kidney. Thus, hTERT is not absolutely required for growth of ALT+ cells beneath the skin.
Evidently, the microenvironment in the kidney is an excellent one 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 (17). This site is also ideal for revealing invasive behavior of tumorigenic cells, as malignant cells grow between the renal tubules, eventually destroying the entire organ (8). Although normal cells, such as normal human adrenocortical cells, form functional vascularized tissue structures under the kidney capsule, they have absolutely no invasive properties with respect to the kidney. A clear boundary with the kidney is maintained and there is no sign of invasion (14–16). A relevant question is whether the kidney is an “ectopic” site for the growth of neoplastic fibroblasts and whether the s.c. site might be more “physiologic”. However, it should be noted that fibroblasts occur in all organs of the body and could potentially form a tumor in any organ. Conversely, s.c. injection typically places the cells beneath the panniculus carnosus and not within the dermis; they are therefore not placed within the skin per se. Further studies are needed to define the cellular and molecular processes/pathways that must be targeted to permit survival and growth of cells in the s.c. 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 s.c. injection experiments should be restudied using growth of cells within an internal organ.
Telomere maintenance mechanisms are needed for the long-term growth of tumors and most cancer cells either express hTERT and are telomerase positive, or have acquired the ability for telomere maintenance via the ALT mechanism (1). The present results indicate that these telomere maintenance mechanisms are equivalent when considered in the context of oncogene cooperation models of human cell tumorigenesis.
Grant support: National Institute on Aging grants AG 12287 and AG 20752 and the Ellison Medical Foundation Senior Scholar Award.
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