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[Cancer Research 64, 6144-6151, September 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Progressive Loss of Malignant Behavior in Telomerase-Negative Tumorigenic Adrenocortical Cells and Restoration of Tumorigenicity by Human Telomerase Reverse Transcriptase

Beicheng Sun1,2, Qin Huang1, Shengxi Liu1, Meizhen Chen1, Christina L. Hawks1, Lishan Wang1, Chuhua Zhang1 and Peter J. Hornsby1

1 Department of Physiology and Sam and Ann Barshop Center for Longevity and Aging Studies, University of Texas Health Science Center, San Antonio, Texas; and 2 Liver Transplantation Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Replicative senescence/crisis is thought to act as a tumor suppressor mechanism. Although recent data indicate that normal human cells cannot be converted into cancer cells without telomerase, the original concept of senescence as a tumor suppressor mechanism is that senescence/crisis would act to limit the growth of telomerase-negative tumors. We show here that this concept is valid when oncogene-expressing human and bovine cells are introduced into immunodeficient mice using tissue reconstruction techniques, as opposed to conventional subcutaneous injection. Primary human and bovine adrenocortical cells were transduced with retroviruses encoding Ha-RasG12V and SV40 large T antigen and transplanted in immunodeficient mice using tissue reconstruction techniques. Transduced cells were fully malignant (invasive and metastatic) in this model. They had negligible telomerase activity both before transplantation and when recovered from tumors. When serially transplanted, tumors showed progressively slower growth, decreased invasion and metastasis, shortened telomeres, and morphological features of crisis. Whereas telomerase was not essential for malignant behavior, expression of human telomerase reverse transcriptase enabled cells from serially transplanted tumors that had ceased growth to reacquire tumorigenicity. Moreover, telomerase-negative oncogene-expressing cells were tumorigenic only when transplanted using tissue reconstruction techniques; human telomerase reverse transcriptase was required for cells to form tumors when cells were injected subcutaneously. This work provides a new model to study crisis in an in vivo setting and its effects on malignancy; despite having invasive and metastatic properties, cells are eventually driven into crisis by proliferation in the absence of a telomere maintenance mechanism.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The development of cancer requires multiple changes in a normal cell. Overcoming the barrier of replicative senescence has often been thought to be essential for cancer formation (1, 2, 3, 4) . Replicative senescence was originally described as a process that limits the growth of normal human cells in culture (4 , 5) . The level of telomerase activity in most normal human cells is insufficient for long-term telomere maintenance; progressive shortening of telomeres leads to telomere dysfunction, resulting in permanent growth arrest, termed senescence or M1 (5, 6, 7) . Cells expressing checkpoint-disrupting proteins such as SV40 large T antigen bypass M1 and eventually enter crisis or M2 (5 , 8) . In crisis, short dysfunctional telomeres cause end-to-end chromosome fusions; in cells with disrupted checkpoints, this results in the following: (a) breakage-fusion-bridge cycles, leading to increasing aneuploidy; and (b) mitotic catastrophe, a failure of cytokinesis, resulting in tetraploidization, multipolar cell division, and gross aberrations in chromosome number (8, 9, 10, 11, 12, 13) . Mitotic catastrophe leads to arrest in mitosis or, alternatively, to the formation of cells with multiple nuclei or a single giant nucleus (12 , 13) . Cells in crisis eventually undergo cell death, but the mechanism is unclear; double-strand breaks, which are characteristic of apoptosis, are infrequent (11 , 14 , 15) . Most cancer cells have activated mechanisms of telomere maintenance, thereby avoiding replicative senescence/crisis, either by expression of human telomerase reverse transcriptase (hTERT) or recombination-based alternative lengthening of telomeres (ALT) (1, 2, 3 , 16) .

There are two models for how replicative senescence/crisis (or low telomerase activity/short telomeres) could act as a tumor suppressor mechanism. In the first model, cells cannot acquire tumorigenic properties without telomerase activity or some other form of telomere maintenance mechanism, independent of telomere length. In the second model, lack of a telomere maintenance mechanism does not prevent acquisition of tumorigenic properties by cells, but telomere shortening eventually acts to limit cell proliferation. Most data in human cells support the first of these possibilities; in most oncogene cooperation experiments, hTERT has been found to be required to convert normal human cells to cancer cells (1 , 2 , 17) . However, the second possibility is supported by the finding that the combination of adenovirus E1A, Ha-RasG12V, and MDM2 did not require hTERT (18) . Oncogene-expressing cells from telomerase-negative mice can also produce tumors. In this model, late-passage cells with short telomeres were still tumorigenic, but they lost the ability to metastasize. However, final cessation of growth of tumors as a result of senescence/crisis did not occur; despite a continuing lack of telomerase activity, telomeres eventually increased in length, and tumor growth was restored (19) . Telomere biology differs substantially between mice and humans (20) . Nevertheless, these experiments show the partial operation of replicative senescence/crisis as a tumor suppressor mechanism that acts to limit malignant properties as telomeres shorten.

Because telomerase activity has been required for tumorigenicity in almost all oncogene cooperation models in human cells, the possibility of observing a reduction in malignant properties as a result of telomere shortening has been precluded. Thus a direct demonstration that replicative senescence/crisis acts as a tumor suppressor mechanism in cells that naturally lack a telomere maintenance mechanism has not been provided. The effects of anti-telomerase and senescence-inducing therapies have been studied in telomerase-positive cancer cells (21, 22, 23, 24) , but these observations are of senescence or crisis acting on cells that are already telomerase positive and therefore do not address the question of how replicative senescence/crisis may act as a mechanism to prevent tumor development. Presumably, if this process acts in the early part of the life span as a mechanism to prevent lethal cancers, it must act to stop the growth of a tumor before cells acquire telomerase activity and indefinite proliferative capacity.

Here we investigated these concepts in a tissue reconstruction model employing primary human and bovine adrenocortical cells (25, 26, 27, 28) . We used two models of tissue reconstruction, cell transplantation under the capsule of the kidney and subcutaneous cell transplantation in collagen gel, both of which have been successfully applied to the experimental formation of human adrenocortical tissue (26 , 29) . We also made extensive use of bovine adrenocortical cells. Like human cells, bovine cells do not have telomerase activity sufficient for telomere maintenance and therefore undergo telomere shortening, leading to senescence (27) . Like human cells, they maintain a stable karyotype under long-term growth in culture, a property most dramatically demonstrated by the production of healthy cloned cattle from late-passage bovine cells (30) . However, they have substantially longer telomeres than human cells (31) , enabling greater cell proliferation in the absence of telomerase, both in cell culture and in tumors.

Tissue reconstruction models differ from conventional tumor assays in immunodeficient mice (subcutaneous and intramuscular injection of cell suspensions) in that cell survival is not severely compromised by the implantation technique. If cell survival is low, as it is in conventional assays, a gene that increases cell survival may appear to be required for tumorigenicity. A tissue reconstruction model has also been used in experimental tumorigenicity studies of human keratinocytes (32) . We introduced cells into the host animal within a very short time after they were transduced with oncogenes, an approach similar to that termed "multiplex serial gene transfer" in the keratinocyte system. In the keratinocyte skin reconstruction model, hTERT was not required for tumor formation, but keratinocytes have sufficient telomerase activity to maintain telomere length; tumors growing in animals did not show telomere shortening, and there was no evidence that cells entered senescence or crisis in tumors. In the present experiments on adrenocortical cells, however, telomerase-negative cells eventually entered crisis in tumors, limiting further expansion, invasion, and metastasis.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Growth of Human and Bovine Adrenocortical Cells in Culture.
Human adrenal glands were obtained from kidney organ donors, and the adrenal cortex was dissociated by enzymatic and mechanical dispersion as described previously (28) . Primary cell suspensions were stored frozen in liquid nitrogen. Frozen cells were thawed and replated in Dulbecco’s modified Eagle’s medium/Ham’s F-12 (1:1) with 10% fetal bovine serum, 10% heat-inactivated horse serum, and 0.1 ng/ml recombinant basic fibroblast growth factor. This was supplemented with 1% (v/v) UltroSer G (Biosepra, Villeneuve-la-Garenne, France), a mixture of growth factors, as described previously. The culture dishes used were coated with type I collagen (Becton Dickinson, Franklin Lakes, NJ). The gas phase used was 90% N2, 5% O2, and 5% CO2.

Bovine adrenocortical cells were derived by enzymatic and mechanical dispersion of tissue from the adrenal glands of 2-year-old steers (25) and grown under the same conditions as human adrenocortical cells, except that standard culture dishes were used.

Retroviral Transduction.
The retroviruses used are of the LX type (33) . Their structures are shown in Table 1Citation . They were constructed from pLEGFP-N1 (BD Biosciences Clontech, Palo Alto, CA) by replacing the neo gene. SV40 large T antigen (TAg) was derived from an intron-less cDNA. The oncogenic Ras used is Ha-RasG12V. One construct contained an internal ribosome entry site (IRES) (34) . Retroviral plasmid pBabe-puro-hTERT was generously donated by J. Campisi (Lawrence Berkeley National Laboratory, Berkeley, CA). The Phoenix cell line (amphotropic) was used for production of retroviral particles (35) . Phoenix cells were transfected with retroviral plasmids by the calcium phosphate method. After 48 hours, the supernatant medium was filtered through a 0.45-µm filter and added to the target cells. Infection was allowed to proceed for 48 h. Because the retroviral constructs used encode green fluorescent protein, the rate of infection of the culture was monitored by fluorescence microscopy. An additional 7 days of growth in culture were used to ensure the impossibility of any infectious virus remaining in the cell population.


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  		Table 1 Tumorigenic conversion of human and bovine adrenocortical cells by RasG12V and SV40 TAg

 
Depending on the experimental protocol used, the population of cells containing a mixture of transduced and nontransduced cells was used for cell transplantation, or alternatively, a pure population of transduced cells was prepared. In the latter case, cells were either cloned by limiting dilution in 96-well plates or sorted by flow cytometry. More than 98% of the cells were fluorescent after sorting. Cloned or sorted cells were expanded in culture for transplantation or biochemical studies. In most cases, no drug selection was used, but some cells (as indicated in the text) were selected with 1 µg/ml puromycin after infection with pBabe-puro-hTERT.

Biochemical Characterization of Retrovirally Transduced Cells.
Western blotting was performed using standard techniques. Antibodies used were as follows: SV40 TAg, mouse monoclonal antibody PAb108 (Santa Cruz Biotechnology, Santa Cruz, CA); and Ras, mouse monoclonal antibody (R02120, Transduction Laboratories, Lexington, KY). Telomerase activity in detergent extracts of cells was assessed by the telomerase repeat amplification protocol (TRAP) assay as described previously (36) . Telomere restriction fragment (TRF) analysis was performed as described previously (27) , except that DNA fragments were separated by pulse-field gel electrophoresis.

Transplantation of Cells in RAG2–/–,{gamma}c–/– and scid Mice.
RAG2–/–, {gamma}c–/– and ICR scid mice were purchased from Taconic (Germantown, NY). Animals (both males and females) at an age greater than 6 weeks (~25 g body weight) were used in these experiments. Procedures were approved by the institutional animal care committee and carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. We transplanted 2 x 106 adrenocortical cells beneath the kidney capsule using the protocol described previously (25 , 28) . Subcutaneous cell transplantation was accomplished by embedding cells in collagen and insertion under the skin as described previously (26) . In these experiments, the cells in collagen were inserted beneath the skin of the external ear. In this site, the growth and vascularization of the tissue or tumor may be monitored in a continuous and noninvasive fashion.1

Animals were sacrificed at various times after transplantation, as described in Results. Tumors were visualized by fluorescence using a 470 nm light source (Lightools Research, Encinitas, CA) 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.

Histological and Immunohistochemical Analysis.
The fixation, paraffin embedding, and histological examination of tissue formed from transplanted cells were carried out using standard techniques. SV40 TAg was detected with monoclonal antibody PAb416 (Oncogene Science, Cambridge, MA). Double-strand breaks characteristic of apoptosis (37) were visualized using the In Situ Oligo Ligation (ISOL) protocol (Chemicon, Temecula, CA) in accordance with the manufacturer’s instructions.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Conversion of Human and Bovine Adrenocortical Cells to a Fully Tumorigenic State by RasG12V and SV40 Large T Antigen.
We used a tissue reconstruction model in which adrenocortical tissue is formed from transplanted primary cells in immunodeficient mice (25, 26, 27, 28, 29) . This model enabled us to investigate the minimal genetic modifications needed for tumorigenic conversion of primary cells, when transplanted using techniques that permit excellent survival of both normal and genetically modified cells. Human and bovine adrenocortical cells were transduced with retroviruses encoding RasG12V and SV40 TAg (Fig. 1Citation ; Table 1Citation ); the SV40 TAg construct is intronless and therefore does not encode small t antigen. The retroviruses that encode both genes in a single construct enabled the transduction of target cells in a single retroviral infection. Cells were then implanted into immunodeficient mice after only a minimal period in cell culture. We hypothesized that this would be especially important in human adrenocortical cells, which have a total replicative potential in the range of 20 to 40 population doublings (38) . It was of less concern in bovine adrenocortical cells, which have a replicative potential of up 100 to 120 population doublings (39) .



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  		Fig. 1. Malignant behavior of human and bovine adrenocortical cells expressing RasG12V and SV40 TAg. Primary human and bovine adrenocortical cells were transduced with RasG12V and SV40 TAg and transplanted beneath the kidney capsule of immunodeficient mice. Images A and B were produced by the use of dual light sources as described in Materials and Methods. A, gross appearance of tumors formed from human adrenocortical cells (left panel) and adrenocortical bovine cells (right panel). B, lung metastases formed from bovine adrenocortical cells. C, detailed view of border with kidney. Top panel, tissue formed from normal, nongenetically modified human adrenocortical cells; note the sharp boundary. Bottom panel, border between kidney and tumor formed from human adrenocortical cells (30 days after cell transplantation); note the irregularity of border and invasion. H&E stain. D, SV40 TAg expression in tumor formed from bovine adrenocortical cells, showing invasion into kidney.

 
Both bovine and human adrenocortical cells were converted to a tumorigenic state by the combination of RasG12V and SV40 TAg. Cells were fully malignant, as evidenced by extensive local invasion and distant metastases (Fig. 1Citation ; Table 1Citation ). Results were similar whether cells were transplanted under the kidney capsule or subcutaneously in collagen gel. The expression of green fluorescent protein in the transplanted cells greatly facilitated gross observations of the extent of growth of the cells and their potential for tumorigenic behavior, such as invasion into host tissue and metastasis. Human cells produced smaller tumors than bovine cells. Nevertheless, tumors from human cells usually showed obvious invasion into the kidney (Fig. 1C)Citation . In contrast, as described previously (29) , tissue formed from nongenetically modified human adrenocortical cells showed a clear boundary with the kidney (Fig. 1C)Citation . Bovine adrenocortical cells produced large tumors that grew progressively in the site of transplantation, invaded neighboring tissues and organs (muscle, fat, and pancreas), and metastasized to the lungs and other organs. Tumors from bovine cells grew very rapidly, often reaching a size of 0.5 cm within 15 to 20 days. Metastases from bovine cells were verified by immunohistochemistry for SV40 TAg. Small lung metastases, detected by fluorescence, were also observed in some animals with human cells, although they were too small to be verified histologically.

The formation of malignant tumors by cells expressing only RasG12V and SV40 TAg was unexpected, in view of the fact that this combination of oncogenes was previously found to be nontumorigenic in several human cell types in the absence of SV40 small t antigen and hTERT (17) . Therefore, we considered the possibility that tumors are formed by a minority cell type within the transduced cell population, i.e., cells that have acquired additional genetic changes after the introduction of RasG12V and SV40 TAg. To test this, we performed a limiting dilution experiment (Table 1)Citation . We found that as few as 100 transduced cells were required to produce a tumor. In view of the fact that the cells were transplanted 7 days after retroviral infection and therefore had only a minimal opportunity for mutations to occur before being introduced into the animal, it seems very unlikely that the malignant potential of the cell population is the result of overgrowth of a minority cell type rather than a property of transduced cells in general.

The much greater replicative potential of bovine cells likely results from their longer telomeres (a TRF length of ~20 kb versus ~12 kb for human cells; ref. 31 ). These properties permitted us to perform some experiments on bovine cells that were not possible with human cells. First, we were able to transplant pure genetically modified cells. Cells were sorted by flow cytometry or clones were isolated by limiting dilution. In all cases, the resultant cell populations formed tumors in immunodeficient mice that were invasive and metastatic (Table 1)Citation . When RasG12V was introduced first, cells grew slowly in culture, as expected (40) , but premature senescence was avoided by the use of 5% oxygen and an antioxidant-rich medium (41) . The order of introduction of the genes, RasG12V and SV40 TAg, did not affect the outcome; both types of clones formed very aggressive tumors.

Serial Growth of Tumors in Host Animals Results in Progressive Loss of Malignant Properties.
Tumors formed from human adrenocortical cells expressing RasG12V and SV40 did not regrow when tumor fragments were retransplanted in 2° host animals, either when fragments were implanted subcutaneously or inserted under the kidney capsule. However, many tumors formed from RasG12V/SV40 TAg-transduced bovine cells could be regrown as tumors in 2° or 3° hosts (Fig. 2)Citation . Not all such tumors could be regrown. Of 20 tumors tested, 9 could be regrown in 2° hosts, and of these, 4 could be regrown in 3° hosts. No tumors could be regrown in 4° host animals, although in two cases tumors reached 0.2 cm before regressing. Similar results were obtained when tumor fragments were implanted subcutaneously or inserted under the kidney capsule. Considering 2° and 3° tumors in aggregate, 40 individual tumor fragments were re-transplanted; none of these yielded a continuously transplantable tumor (they either failed to grow or grew and regressed).



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  		Fig. 2. Progressive decrease in tumor growth in serial transplantation. A, animals with tumors formed from retransplanted tumor fragments. From left to right: 2° host animal (20 days) with tumors from fragments of a 1° tumor (derived from bovine adrenocortical cells expressing RasG12V and SV40 TAg); 3° host animal (42 days) with tumors from fragments of the 2° tumor; and 4° host animal bearing fragments from the 3° tumor, which enlarged only to <0.2 cm and subsequently regressed. B–D, histological appearance of 1° (B)-, 2° (C)-, and 3° (D) tumors; trichrome stain; connective tissue is blue, muscle is bright red.

 
A striking loss of growth rate was observed in the 3° tumors and in those 2° tumors that could not be transplanted further. These terminal tumors required >45 days to reach 0.5 cm and then did not show further enlargement over the next 30 days. In contrast, the 1° tumors from which these tumors were derived grew continuously to a size that necessitated the sacrifice of the host animals.

The reduction in growth rate of 2° and 3° tumors was accompanied by loss of malignant behavior. 1° tumors, whether originating in the kidney or subcutaneously from collagen gel transplants, extensively invaded neighboring tissues. Fig. 2BCitation shows histology of a 1° tumor transplanted subcutaneously in collagen gel. Second- and 3° tumors derived from this 1° tumor (Fig. 2C and D)Citation showed much less invasion into the surrounding tissue (e.g., muscle), and the 3° tumor became encapsulated with connective tissue. 2° and 3° tumors usually showed central necrosis.

Tumor cell fluorescence enables the detection of very small metastases. Although metastases to the lungs were frequent in animals with primary tumors (Table 1)Citation , we found no lung metastases in any animals with 2°- or 3° tumors (0 of 13 animals).

The Cessation of Growth in Tumors Is Accompanied by Crisis.
In culture, the presence of SV40 TAg causes cells to bypass M1/senescence and eventually reach M2/crisis. We hypothesized that the cause of the slower growth and loss of malignant properties is the result of crisis in tumors. Whereas cells in 1° tumors formed from RasG12V/SV40 TAg-transduced bovine cells were predominantly of a uniform size and showed unremarkable nuclear morphology (Fig. 3A)Citation , in 2° and particularly in 3° tumors, cells and nuclei became much more heterogeneous (Fig. 3B)Citation . Large areas of 3° tumors comprised cells with enlarged and irregularly shaped nuclei, atypical mitotic chromosomes, or condensed chromatin masses (Fig. 3C)Citation . Although very few normal-appearing mitoses were observed in 3° tumors, many cells were positive for the proliferation marker Ki-67. Anaphase bridges were observed in 1° and 2° tumors (Fig. 3D)Citation but were rarely observed in 3° tumors. These data are consistent with the progressive entry of cells into crisis during growth in 1° and 2° tumors (8, 9, 10, 11, 12, 13) . In 3° tumors, end-stage crisis is observed as the presence of large numbers of abnormal cells that are not proliferatively quiescent yet are unable to complete normal cell division.



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  		Fig. 3. Crisis in tumors formed from RasG12V/SV40 TAg-expressing cells. A and B, H&E-stained sections of 1° (A)- and 2° (B) tumors formed from RasG12V/SV40 TAg-expressing bovine adrenocortical cells. C, examples of abnormal mitotic chromosomes and a nuclear bridge in 3° tumors. D, examples of anaphase bridges found in 1° and 2° tumors.

 
Because human cells forming tumors were unable to be regrown in secondary hosts, we hypothesized that human cells may have already entered crisis in primary tumors. In 1° human tumors, many areas comprised cells with abnormal appearance similar to that of 3° bovine tumors.

Although there was central necrosis in 3° bovine tumors, outside of those regions we detected rather few cells with DNA double-strand breaks, which are characteristic of cells undergoing apoptotic cell death (37) . Fewer than 1% of cells with typical apoptotic chromatin condensation that were positive for double-strand breaks (ISOL+) were observed. Cells with enlarged nuclei or abnormal mitotic chromosomes were ISOL–.

Telomerase Activity and Telomere Length in RasG12V/SV40 Large T Antigen-Transduced Cells.
The progressive decrease in malignant properties seen in serially transplanted tumors formed from RasG12V/SV40 TAg-expressing cells appeared to be the result of crisis in the tumor cells. We hypothesized that cells are unable to maintain telomeres and that the resultant telomere dysfunction causes end-to-end fusions, as evidenced by anaphase bridges, which in turn result in mitotic catastrophe, seen as the accumulation of cells with bizarre nuclear morphology and abnormal mitotic chromosomes (8, 9, 10, 11, 12, 13) . Consistent with this scenario, RasG12V/SV40 TAg-expressing human and bovine adrenocortical cells had very low levels of telomerase activity (ref. 36 ; Fig. 4Citation ). Cells isolated from bovine tumors and sorted by flow cytometry to remove contaminating mouse cells also had negligible levels of telomerase activity. Telomerase activity was assessed in cells transduced with each of the various retroviral constructs used in these experiments (Table 1)Citation and was negligible in all cases. Thus differences among the constructs observed as rate of growth and tendency for invasion and metastasis (Table 1)Citation were unlikely to result from variations in telomerase. A lower degree of malignant behavior may result from a lower level of Ras in cells in which the gene is expressed from an IRES (Fig. 4)Citation .



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  		Fig. 4. Biochemical characteristics of adrenocortical cells transduced with RasG12V and SV40 TAg. Top panel, TRAP assays; telomerase activity was assessed in samples containing 50, 100, and 200 ng of protein. 0, no sample; 1, bovine adrenocortical cells transduced with hTERT; 2, bovine adrenocortical cells transduced with RasG12V and SV40 TAg, before transplantation; 3, bovine adrenocortical cells transduced with RasG12V and SV40 TAg, isolated from a tumor; 4, human adrenocortical cells, not transduced; 5, human adrenocortical cells transduced with RasG12V and SV40 TAg; 6, human adrenocortical cells transduced with RasG12V, SV40 Tag, and hTERT; 7, 3T3 cells (200 ng). i.c., internal control. Bottom left panel, telomere restriction fragment analysis of DNA from tumors and their precursor cells. Bovine adrenocortical cells (Lane 1) were transduced with SV40 TAg only and cloned (Lane 2) or transduced with RasG12V and then with SV40 TAg and cloned (Lane 3). The latter cells were transplanted into a mouse and formed a tumor (Lane 4); a fragment of this tumor was retransplanted to form a 2° tumor (Lane 5). These tumor cells were isolated and transduced with hTERT in culture (Lane 6). They were then implanted into a mouse and formed a tumor (Lane 7) that was serially transplanted into 2°, 3°, and 4° host animals (Lanes 8–10). Bottom right panel, Western blots for Ras, SV40 TAg, and ß-actin. Lane 1, bovine adrenocortical cells transduced 1° with RasG12V and then with SV40 TAg, before transplantation; Lanes 2 and 3, cells isolated from 1° and 2° tumors derived from these cells; Lane 4, cells transduced 1° with SV40 TAg and then with RasG12V; Lane 5, cells transduced with retrovirus LTR-Ras-CMV-SV40T; Lane 6, cells transduced with retrovirus LTR-SV40T-IRES-Ras; Lane 7, nontransduced cells.

 
Because it was not possible to flow-sort sufficient cells from human tumors, which were smaller than bovine tumors, we examined the expression of hTERT mRNA by real-time PCR on RNA isolated directly from tumors. Whereas hTERT was detectable at high cycle numbers in tumors, the level was not higher than that seen in cells before cell transplantation, which were negative in the TRAP assay.

TRF length was analyzed in flow-sorted cells from bovine tumors. In primary cells, TRF was ~20 kb, as reported previously (27 , 31) . After transduction with RasG12V and/or SV40 TAg, TRF decreased (Fig. 4)Citation . In cells isolated from tumors, the telomeric DNA signal was extremely weak and difficult to distinguish from background. The strong reduction in signal resembled that observed previously in senescent nongenetically modified bovine adrenocortical cells (27) .

Restoration of Tumorigenicity by Transduction with Human Telomerase Reverse Transcriptase.
We hypothesized that the entry of cells into crisis in tumors is caused by telomere shortening; initially, RasG12V and SV40 TAg drive proliferation, invasion, and metastasis, but as cells enter crisis, these properties are progressively lost. If this model is correct, then it should be possible to restore tumorigenicity to cells by introduction of hTERT, thereby restoring telomerase and telomere maintenance. Cells were isolated from tumors and returned to cell culture. Cells from 2° or 3° tumors derived from bovine cells and from 1° tumors derived from human cells grew poorly in culture and exhibited typical signs of crisis. As usually observed for cells in crisis, there was cell division, but the overall population size progressively decreased because of detachment of cells from the substratum. We did not observe escape from crisis in either human or bovine tumor cells. However, when tumor cells were transduced with hTERT, telomerase activity increased to high levels, the cell growth rate increased, and the cells proliferated indefinitely in culture.

Cells isolated from 2° or 3° tumors derived from bovine cells and from 1° tumors derived from human cells did not form tumors when retransplanted. However, after transduction with hTERT, tumorigenicity was restored (Fig. 5)Citation . Cells formed tumors either when injected under the kidney capsule or when implanted subcutaneously in collagen gel. Whereas tumors formed from RasG12V/SV40 TAg-transduced cells could not be serially transplanted beyond the 3° host, tumors formed from the same cells after transduction with hTERT could be transplanted at least to 4° hosts. Moreover, the tumors showed a progressive increase in growth rate in successive hosts (Fig. 5)Citation . The increased rate of growth was accompanied by a progressive increase in TRF length (Fig. 4)Citation , an observation consistent with the concept that the restoration of a telomere maintenance mechanism is responsible for the restoration of tumorigenicity.



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  		Fig. 5. Restoration of tumorigenicity by hTERT. Representative experiments are shown in which cells isolated from telomerase-negative tumors formed from RasG12V/SV40 TAg-transduced human and bovine adrenocortical cells were transduced with hTERT and retransplanted into immunodeficient mice. After establishment of the 1° tumor, 2° and subsequent tumors were formed by subcutaneous implantation of tumor fragments. The retroviruses used are indicated. The times shown are the number of days after transplantation before the tumor reached a diameter of 0.5 cm. The numbers of animals used are indicated in parentheses.

 
Tumorigenicity of Oncogene-Expressing Cells after Subcutaneous Injection: Requirement for Human Telomerase Reverse Transcriptase.
Cells transduced with hTERT as well as RasG12V and SV40 TAg produced tumors when injected subcutaneously as cell suspensions, the conventional manner for assessment of tumorigenicity. We used a population of RasG12V/SV40 TAg-transduced bovine adrenocortical cells that produced aggressive tumors when implanted by tissue reconstruction methods (Fig. 1Citation ; Ras->SV40 TAg in Table 1Citation ). Aliquots of these cells were injected under the skin of immunodeficient mice, or alternatively, these cells were first transduced with hTERT, selected by drug resistance, and then injected under the skin. In 10 of 10 individual injections of 2 x 106 cells each, RasG12V/SV40 TAg/hTERT-transduced cells produced tumors that expanded continuously and invaded adjacent tissues (Fig. 6)Citation . However, RasG12V/SV40 TAg-transduced cells without hTERT did not produce tumors when injected subcutaneously. Small fluorescent nodules (<0.2 cm in diameter) could be observed immediately after injection. Although these fluorescent nodules could sometimes be observed for as long as 30 days, they did not enlarge and eventually became undetectable. In a total of 10 individual injections, no RasG12V/SV40 TAg-transduced cells grew beyond 0.2 cm, and all eventually disappeared.



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  		Fig. 6. Requirement for hTERT for tumorigenicity when cell suspensions are injected subcutaneously. A, RasG12V/SV40 TAg-transduced bovine adrenocortical cells were injected under the skin at four points on the back of an immunodeficient mouse. Immediately after injection, small fluorescent nodules were visible (left panel). An animal that received four injections of these cells was photographed after 20 and 30 days (middle and right panels). B, an animal that received injections of RasG12V/SV40 TAg-transduced cells that had additionally been transduced with hTERT was photographed at 14 (left panel), 20 (middle panel), and 25 days (right panel).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The concept that replicative senescence can act as a tumor suppressor mechanism was proposed by Ruth Sager (42) in the 1980s. This hypothesis was based on the resistance of human cells to oncogenic transformation and the observation that cells transfected with SV40 T antigen and v-Ki-Ras could form tumors that regressed after reaching 1 cm in size (42) . However, the basis for regression was not determined. It was not clear whether crisis or senescence occurred in the tumors, and if so, whether this changed malignant properties in terms of growth rate, invasion, and metastasis. Subsequently, the phenomenon of experimental tumors that ceased growth or grew transiently and regressed was not widely studied, perhaps because of the widespread use of immortalized cell lines as starting material for oncogene studies in tumorigenesis.

Here we demonstrate that cells in telomerase-negative tumors formed from human and bovine adrenocortical cells expressing RasG12V and SV40 large T antigen accumulate in crisis. This state is accompanied by loss of invasion and metastasis and by a tendency for tumors to become encapsulated. Normal mitoses are rare, but cells appear to be in the cell cycle. There is a remarkable heterogeneity of cell morphology. Many cells have giant nuclei, multiple nuclei, or abnormal mitotic chromosomes. Karyotypes in cells from tumors show multiple abnormalities, whereas they are normal in oncogene-transduced cells at the time of cell transplantation (data not shown). The restoration of tumorigenicity in these cells by introduction of hTERT confirms that the cause of the progressive loss of tumorigenic properties is the lack of a telomere maintenance mechanism. Thus, these data clarify the role of telomerase: in this model it is not required for tumorigenicity (including invasion and metastasis), but it is required to permit continued growth of tumors when telomere shortening proceeds to a critical extent.

The novel observation of crisis in telomerase-negative malignant tumors raises several questions. First, although our tests were limited, crisis acted as a reliable tumor suppressor mechanism. No tumors showed evidence of escape from crisis, such as focal regrowths, which could have been caused by cells activating telomerase or ALT (1, 2, 3 , 16) . In this respect, human and bovine adrenocortical cells may differ from telomerase-negative tumorigenic mouse cells, which apparently became ALT+ (19) . Second, there was little evidence of cell death, except in the central necrotic area of the tumor. A few cells with apoptotic features were observed. In culture, cells in crisis have some features of apoptotic cells yet show little evidence of DNA breakage (11 , 14 , 15) . It is possible that cell death in culture is a nonspecific result of detachment from the substratum. Therefore, the possibility should be considered that crisis does not directly lead to cell death in the in vivo environment. Cells that have failed to undergo cytokinesis after mitosis may become resistant to apoptosis (43) . Third, an important question is whether the operation of telomere shortening and crisis, of the type observed in these experiments, would act to suppress incipient human tumors under normal circumstances. We observed that tumorigenic telomerase-negative human cells could form tumors up to 1 cm in diameter, at which point they ceased growth and had histological features of crisis. Such a tumor in the human body might go undetected under many circumstances. Although the majority of cancers appear to have a telomere maintenance mechanism, some early cancers do not (15 , 44) . The fate of such tumors, if they remained untreated, is unclear.

Fourth, crisis in telomerase-negative tumors differs in significant ways from the response of telomerase-positive cancers to chemotherapy, including therapy with anti-telomerase agents (21 , 22) . Importantly, anti-telomerase therapy may in fact induce ALT in tumor cells (45) . Many chemotherapeutic agents cause premature senescence in tumors, which is independent of telomerase activity and telomere length (23 , 24 , 46) . Therefore, a pure effect of such agents to cause crisis, of the type observed here in telomerase-negative tumors, is not expected.

The use of tissue reconstruction methodology yields conclusions that conflict with those obtained from studies that used more conventional methods for selection of transduced cells and assessment of tumorigenicity in immunodeficient mice (1 , 2 , 17) . Even in the adrenocortical tissue reconstruction model we had previously concluded that hTERT was required for tumorigenicity (27) . In that case, genetic modifications (Ras and SV40 TAg) were introduced into cells sequentially with successive drug selections. This may have either exhausted too much of the replicative potential of the cells or inflicted nonlethal damage of other kinds, resulting in an apparent requirement for hTERT for tumor formation. The latter possibility is supported by other evidence that the requirement for hTERT may not be linked to telomere maintenance. In an ALT+ cell line, SV40 TAg and Ras were insufficient for tumorigenicity, and hTERT was required (47) . We also found in these experiments that hTERT was required for tumor formation when cells were injected subcutaneously (the method used for tumorigenicity testing in the majority of studies of oncogene cooperation in human cells), rather than transplanted using tissue reconstruction methods. Taken together, these results suggest that an activity of hTERT other than telomere length maintenance, such as resistance to apoptosis (48) , may be responsible for the apparent absolute requirement for hTERT for tumor formation.


   FOOTNOTES
 
Grant support: Grants AG 12287 and AG 20752 from the National Institute on Aging and by a Senior Scholar Award from the Ellison Medical Foundation.

Note: Present address for M. Chen, Department of Pathology, Shantou University Medical College, Shantou, China.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Peter J. Hornsby, University of Texas Health Science Center, 15355 Lambda Drive STCBM 3.100, San Antonio, TX 78245. Phone: 210-562-5080; Fax: 281-582-3538; E-mail: hornsby{at}uthscsa.edu

1 B. Sun and P. Hornsby, unpublished observations. Back

Received 4/26/04. Revised 6/19/04. Accepted 6/30/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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