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Cell Growth & Differentiation Vol. 13, 397-407, September 2002
© 2002 American Association for Cancer Research

Malignant Transformation in Human Chondrosarcoma Cells Supported by Telomerase Activation and Tumor Suppressor Inactivation1

James A. Martin2, Erin Forest, Joel A. Block, Aloysius J. Klingelhutz, Brent Whited, Steven Gitelis, Andrew Wilkey and Joseph A. Buckwalter

Departments of Orthopaedic Surgery [J. A. M., E. F., B. W., A. W., J. A. B.] and Microbiology [A. J. K.], The University of Iowa, Iowa City, Iowa 52242, and Section of Rheumatology, Department of Internal Medicine [J. B.], Section of Medical Oncology, Department of Orthopedics [S. G.], Rush-Presbyterian-St Luke’s Medical Center, Chicago, Illinois, 60612


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Human chondrosarcomas do not respond to current chemotherapies or radiation therapy, and their size and histological appearance do not reliably predict the risk of local recurrence and metastases, making selection of surgical treatment difficult. Identifying mechanisms responsible for the proliferation and invasive behavior of these tumors would be of immense clinical value. We hypothesized that telomerase expression is one of these mechanisms. We detected telomerase expression in 7 of 16 chondrosarcomas, but cells cultured from telomerase-negative chondrosarcomas acquired strong telomerase activity and lost tumor suppressor activity after their establishment in culture. These changes were associated with accelerated indefinite cell proliferation, morphological transition, and increased invasive activity, indicating that telomerase activation and loss of cell cycle control leads to the emergence of aggressive cells from chondrosarcoma cell populations. These observations may lead to better understanding of the factors responsible for malignant transformation, local recurrence, and metastases of cartilage neoplasms.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Human chondrosarcomas present uniquely difficult clinical problems. These poorly understood malignant neoplasms of cartilage-forming cells vary considerably in clinical presentation and biological behavior. In many instances, they originate in benign cartilage neoplasms, enchondromas and osteochondromas, that have been present for years and that in most individuals follow a benign course for a lifetime. Some chondrosarcomas grow slowly for many years and, although they may become large enough to cause disability because of their physical bulk, they never spread from their original site, nor do they invade surrounding normal tissues. Other chondrosarcomas aggressively enlarge, invade, and destroy normal tissue and rapidly spread throughout the body, leading to death (1) . Chondrosarcomas that have grown slowly for years and benign cartilage neoplasms, enchondromas and osteochondromas, can become extremely aggressive without any apparent inciting event. The behavior of these tumors is extremely difficult to predict from evaluation of their size and histological appearance, and multiple attempts to develop better methods of evaluating their potential for local tissue invasion and metastatic spread have failed (2) . Unfortunately, conventional treatments for malignancy, such as the wide range of current chemotherapy drugs and radiation therapy, are generally ineffective in the treatment of chondrosarcomas (3) . Partially because these tumors are rare and partially because their biological behavior differs so much from other malignancies, there has been relatively little basic investigation that would help form the basis for improved therapies. Recent progress includes the finding that resistance to chemotherapy may be attributable, in part, to multidrug resistance-1 and to P-glycoprotein expression (4) . Aberrant oncogene expression (5, 6, 7) and molecular lesions in the p16INK4a/pRb pathway may also be characteristic of chondrosarcomas (8, 9, 10, 11) . These observations represent significant advances in our understanding growth controls in the human chondrosarcomas, but it is not yet clear how such changes contribute to malignancy.

Cellular immortalization is believed to be necessary for malignant progression. Thus, knowledge of the molecular pathways that lead to immortalization may lead to the identification of useful prognostic markers and provide a basis for therapeutic interventions designed to arrest tumor growth (12 , 13) . Cancer cell immortality is often associated with the activation of telomerase, a polymerase that catalyzes telomere elongation (14, 15, 16, 17) . Although it is not expressed in most normal somatic cells, biopsy studies indicate telomerase activity is present in the majority of malignant tumors. For example, telomerase is expressed in ~80% of infiltrating and in situ breast carcinomas and is associated with tumor size, lymph node metastases, and poor prognosis (18, 19, 20) . Telomerase expression is also associated with poor prognosis in such diverse cancers as thyroid cancer (21) , adrenocortical cancers (22) , in pheochromocytomas (23) , neuroblastomas (24) , prostate cancers (25) , lung cancers (26) , and meningeal cancers (27) . These data indicate that telomerase activity levels correlate with outcome in a wide variety of human cancers and suggest that telomerase inhibitors may prove useful in blocking tumor progression. However, telomerase activity is also detected in premalignant prostate (28) and breast neoplasias (29) , suggesting that it is not always associated with overt malignancy. Moreover, ~10% of all malignancies are known to maintain telomere length via a telomerase-independent pathway, a recombination-based alternative (ALT) mechanism of telomere maintenance (30, 31, 32) . The ALT mechanism appears to be active in high-grade osteosarcomas that show a relatively low incidence of telomerase activity (52%) and chromosomal abnormalities associated with ALT-induced recombination (31, 32, 33) . These data indicate that a substantial subset of tumors would lack sensitivity to many telomerase-specific inhibitors.

Comparatively little work has been done to determine the mechanisms of telomere maintenance in the human chondrosarcoma. One recent attempt failed to detect telomerase activity in 29 chondrosarcoma biopsies using a standard activity assay; however, the negative results might have been attributable to the presence of telomerase assay inhibitors in the chondrosarcoma extracts used in this study (34) . In contrast, another study showed telomerase expression in tumor tissue by both the activity assay and real-time PCR analysis for hTERT,3 the limiting component of telomerase (33) . These findings indicated that telomerase was expressed in ~80% (five of six) of Grade I tumors and 100% (four of four) of grade III tumors. Analysis of a small set of chondrosarcomas (n = 5) by Sangiorgi et al. (35) showed an apparent correlation between high telomerase activity and recurrence or metastasis. Although these results indicate that telomerase is prevalent in chondrosarcomas, the relationship between tumor cell proliferation and telomerase expression was not investigated; thus, the biological significance of telomerase expression in chondrosarcomas remains unknown. With these issues in mind, we sought to confirm the apparently high incidence of telomerase activity in chondrosarcomas and to determine whether there is an association between telomerase activity and the growth characteristics of chondrosarcoma cell populations. Telomerase activity and telomere length were measured in cell populations derived from 11 chondrosarcomas and 2 enchondromas. Population growth rates and telomere dynamics during serial subcultivation were assessed in 3 chondrosarcoma-derived cell lines that did not initially express telomerase and in 1 cell line that did express telomerase.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Relevant clinical data regarding the sources of the cell lines used in this study are shown (Table 1)Citation . Cell lines were derived from tumors that varied in histological grade from I to III. Although long-term follow-up data were not available for all cases, metastasis or local recurrence within 1 year of initial diagnosis was included as an indication of tumor aggressiveness. The JJ, FS, BG, and 105 cell lines were isolated and characterized previously (10 , 11 , 36 , 37) . Cultures of these lines were established from frozen stocks. The CS and EN cell lines were started immediately after their isolation from tumors.


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Table 1 Origins of Chondrosarcoma Cell Lines

 
Initial Telomerase Activity and Telomere Length.
RTA and MTL are plotted together to show the relationships between these parameters within 7 days of starting the cultures (Fig. 1)Citation . Data are shown for 16 chondrosarcomas, 2 enchondromas, an hTERT-transduced chondrocyte line (positive control), and a normal chondrocyte population (negative control). A previously published study showed that the RTA level in the hTERT line (1.9 units) was sufficient to maintain telomere length at >12 kbp for >120 PD (38) . RTA in 3 of the chondrosarcoma lines was greater than in hTERT cells (JJ, 2.6 units; CS-6, 4.7 units; and CS-8, 2.4 units), and activity in two additional lines was near the hTERT level (FS, 1.5 units; and CS-9, 1.7 units). Substantially lower activity levels (10–25% of the hTERT line) were found in the CS-11 line (0.2 units) and CS-13 line (0.4 units). Activity in several additional cell lines was 10% or less of the hTERT cell activity (CS-3, 0.08 units; CS-4, 0.09 units; CS-7, 0.16 units; CS-10, 0.10 units; and CS-12, 0.1 units). Background activity (<1.0% of the hTERT line) was found in the remaining chondrosarcoma cell lines (4BG, 0.003 units; 105, 0.015 units; CS-2, 0.009 units; and CS-5, 0.045 units), normal chondrocytes (0.030 units), and enchondroma lines (EN-1, 0.007 units; and EN-2, 0.070 units). PCR products from these TRAP assays were analyzed by polyacrylamide electrophoresis and Southern blotting (Fig. 2)Citation . Negative controls consisting of PCR reactions done with heat-inactivated extracts were assayed for each cell line. Additional controls included the manufacturer’s positive control extract (CON+) and a negative control consisting of PCR reactions performed with lysate buffer in place of cell extract (CON-). Signals for the internal standard appeared in all reactions near the top of the gel (~260 bp), and signals for the unextended telomerase template appeared in all reactions near the bottom of the gel (52 bp). Between these upper and lower bands are 6-bp ladders representing telomerase-catalyzed extensions of the template. Ladders were apparent for the positive control extract and for all of the lines that showed strong telomerase activity in the colorimetric ELISA assay (JJ, FS, CS-6, CS-8, CS-9, hTERT, and CON+). The intensities of these signals were dramatically reduced in heat-treated controls. Relatively faint ladders composed of short extension products were also occasionally observed in lines that showed weak activity in the ELISA assay. These results support the validity of the ELISA-based TRAP assay as an instrument for measuring telomerase activity in chondrosarcoma cell lines.



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Fig. 1. Telomere length and telomerase activity in chondrosarcoma cell lines. Initial mean telomere length (columns) and relative telomerase activity (boxes +/- standard deviations) in chondrosarcoma cell lines (JJ, FS, 4BG, 105, CS-2, CS-3, CS-4, CS-5, CS-6, CS-7, CS-8, CS-9, CS-10, CS-11, CS-12, and CS-13), enchondroma cell lines (EN-1 and EN-2), in normal chondrocytes (CH), and in chondrocytes transduced with the human telomerase gene (hTERT) are shown. Bars, SD.

 


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Fig. 2. Polyacrylamide gel analysis of TRAP assay products. Display of TRAP assay PCR products from early cultures of the cell lines shown in Fig. 1Citation . Results for untreated (+) and heat denatured lysates (-) are shown for each cell line. Control lanes (CON) show products generated from the manufacturer’s positive control template (+) and from a lysate buffer blank (-). Six-nucleotide ladders generated by telomerase activity are apparent in the JJ, FS, hTERT, CS-6, CS8, CS-9, and CON lanes.

 
Telomere lengths in chondrosarcoma and enchondroma cell lines were variable with a mean of 7.6 kbp ± 2.1 kbp (SD), comparable with published values for primary human chondrocytes (39) . The maximum length was 13.0 kbp in the hTERT-transduced cell line, and the minimum was 4.0 kbp in the EN-1 cell line. MTL in the chondrosarcoma cell lines did not appear to correlate with telomerase activity; the mean for telomerase-positive lines (7.8 kbp) was not significantly different from the mean for telomerase-negative lines (7.4 kbp; P = 0.66). In contrast, the average MTL for the FS, JJ, BG, and 105 (mean, 5.0 kbp) was significantly smaller than the average MTL for the CS lines (mean, 8.3 kbp; P = 0.002).

In Vitro Growth of Telomerase-negative and Telomerase-positive Chondrosarcoma Cell Lines.
The CS-2, CS-3, CS-4, and CS-8 cell lines were serially subcultured to investigate population growth potential and telomere dynamics during growth. Population growth rate determinations were based on plots of PD versus time (Fig. 3)Citation . A distinct transition in growth rate was apparent in plots of the early phase of growth for the CS-2, CS-3, and CS-4 cell lines (Fig. 3A)Citation . Growth slowed dramatically, reaching a plateau at ~20 days of culture (or after 5–12 PDs) but resumed shortly thereafter. For the CS-2, CS-3, and CS-4 lines, growth continued at a constant rate to at least 100 PDs. Regression analysis of the growth plots revealed that the mean growth rate for the first 5–12 PDs (0.44 ± 0.10 PDs/day; r = 0.98; P = 0.02) was significantly slower than the mean growth rate after 5–12 PDs (0.82 ± 0.03 PDs/day; r = 0.99; P < 0.0001). The CS-8 line did not reach a distinct plateau but did grow more slowly before 5 PDs than after 5 PDs. The increased rate persisted to >60 PD (Fig. 3B)Citation . Unlike the CS lines, the FS, JJ, BG, and 105 cells lines grew at a constant rate for at least 80 PDs (Fig. 4)Citation . In contrast, a normal chondrocyte population grew at constant rate for ~25 PDs but stopped growing after this point (Fig. 4B)Citation . Because all of the lines were cultured under the same conditions, these data indicate that the observed changes in the growth rates of CS lines were not attributable to general incubation conditions during the experiment.



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Fig. 3. Growth rates of CS-2, CS-3, CS-4, and CS-8 cell lines. A, PD versus time in culture from 0 to 40 days of culture. The arrow points to a period when growth rates reached a brief plateau. Regression analysis of growth prior to the plateau is indicated by the solid line, and analysis of postplateau growth is indicated by the dashed line. Slopes and regression parameters for the two analyses are indicated. B, PD versus time in culture from 0 to150 days of culture.

 


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Fig. 4. Growth rates of FS, JJ, BG, and 105 chondrosarcoma cell lines and a normal chondrocyte population. PD versus time in culture is shown. A, 0–50 days of culture for the FS, JJ, BG, and 105 lines. B, 0–120 days of culture for the FS, JJ, BG, and 105 lines and a normal chondrocyte population (NC).

 
Growth-related Changes in MTL and Telomerase Activity.
MTL was measured at intervals during serial subcultivation of the CS-2, CS-3, CS-4, and CS-8 cell lines. These data were plotted as a function of PDs (Fig. 5)Citation . In the initially telomerase-negative CS-2, CS-3, and CS-4 lines, average initial MTL declined for a time in culture. Initial average MTL (9.2 kbp) was significantly greater than average MTL measured near 20 PDs (6.7 kbp; P = 0.041). In the case of the CS-2 line, MTL declined from an initial length of 9.4 to 6.8 kbp over 19.5 PDs, a rate of telomere erosion equal to 0.133 kbp/PD. MTL in the CS-3 line was 10.3 kbp initially and declined to 7.5 kbp by 22 PDs, an erosion rate of 0.123 kbp/PD. In the CS-4 line, MTL declined from 8.0 to 5.2 kbp over 19 PDs, a rate of 0.146 kbp/PD. After the first ~20 PD, shortened telomeres were maintained for an additional 80 PDs of growth. In contrast, MTL in the CS-8 (telomerase positive) line was relatively constant, declining slightly from 9.5 to 8.6 kbp over 20 PDs (0.045 kbp/PD) and rebounding to 9.2 kbp by 35 PDs.



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Fig. 5. Telomere erosion during serial subcultivation of chondrosarcoma cell lines. MTLs versus PDs for the CS-2, CS-3, CS-4 and CS-8 cell lines are shown.

 
RTA in the CS-2, CS-3, and CS-4 cell lines was assayed to determine whether the arrest of telomere erosion was associated with an increase in telomerase activity. The CS-8 line was included in this analysis to determine whether the telomere length stability we observed in these cells was associated with continuous telomerase activity. RTA was measured at successive passages of each cell line and was plotted as a function of PD (Fig. 6)Citation . These data showed marked increases in RTA from <0.1 unit to >1.5 units in the CS-2, CS-3, and CS-4 lines, coinciding with stabilization of telomere length. Significant activity (>1 RTA unit) was maintained in all subsequent cultures assayed. In contrast, telomerase activity in the CS-8 line remained relatively constant at ~2.0 units from 0 to near 40 PDs.



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Fig. 6. Telomerase activity during serial subcultivation of chondrosarcoma cell lines. RTAs versus PDs for the CS-2, CS-3, and CS-4 cell lines are shown.

 
Growth-related Changes in Tumor Suppressor Protein Expression.
Western blot analysis was used to determine whether time-dependent changes in growth rates and cell morphology were accompanied by loss of tumor suppressor protein expression (Fig. 7)Citation . Side-by-side comparisons of early and late cultures of the CS-2 line (2 PDs versus 106 PDs), the CS-3 line (2 PDs versus 59 PDs), the CS-4 line (2 PDs versus 58 PDs), and the CS-8 line (2 PDs versus 43 PDs) revealed a striking decrease in p16 INK4a expression. p16 INK4a was detectable in early cultures when 10 µg of total protein were loaded but was undetectable in later cultures of the same cell lines, even when 20 µg of total protein was loaded. That there were no PD-related changes in expression of p21waf pRb was detected in both early and late cultures, but there was a change in the banding pattern with time; a single band was predominant in early cultures, but an additional band of slightly higher molecular weight appeared in late cultures. These results suggest that pRb in early cultures was primarily in a hypophosphorylated (active) state, whereas pRb in late cultures is present in both hypo- and hyperphosphorylated (inactive) states. There was also a striking increase in p53 expression in late versus early cultures and a slight decrease in p21waf in late versus early cultures.



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Fig. 7. Tumor suppressor protein levels in early- and late-passaged chondrosarcoma cells. Side-by-side comparisons of early (E) and late (L) cultures of the CS lines are shown. CS-2 cells were analyzed at 2 and 106 PDs; CS-3 cells at 2 and 59 PDs; CS-4 cells at 2 and 58 PDs; and CS-8 cells at 2 and 43 PDs. Ten µg of total protein were loaded in all lanes, except for the p16ink4a late-passaged lanes, which were loaded with 20 µg of total protein. The hypophosphorylated (o) and hyperphosphorylated (p) forms of pRb are indicated.

 
Growth-related Changes in Morphology and Invasive Activity.
Phase-contrast microscopy revealed changes in cell morphology during serial subculture of the CS-2, CS-3, CS-4, and CS-8 cell lines (Fig. 8)Citation . Smaller, more refractile cells emerged initially as islands in a background of fibroblastic-appearing cells and became predominant in subsequent cultures. This change occurred over two to three passages and was complete by 20–25 PDs. The morphology of the FS, BG, 105, and JJ lines did not change with time (data not shown).



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Fig. 8. Morphological changes during serial subculture of chondrosarcoma cells. Phase-contrast micrographs showing cellular morphology before and after 20 PDs are shown. CS-2 (left, 2 PDs; right, 25 PDs); CS-3 (left, 5 PDs; right, 27 PDs); CS-4 (left, 2 PDs; right, 24 PDs); CS-8 (left, 1 PD; right, 43 PDs). Bar (lower right), 100 µm.

 
The invasive activity of the CS lines also changed with time in culture. Invasive activity was compared at the same early and late time points used for Western blot analysis. A population of normal chondrocytes was included as a negative control (Fig. 9)Citation . Activity ranged from a high of 44% (late CS-2 culture) to a low of 2% (normal chondrocytes). Comparisons between early and late cultures showed significant increases in invasion activity for the CS-2, CS-3, and CS-4 cell lines (P < 0.001) but not for the CS-8 line (P = 0.36) or for normal chondrocytes (P = 0.57).



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Fig. 9. Invasive activity in early- and late-passaged chondrosarcoma cells. A, a typical set of micrographs showing CS-3 cells on invasion assay membranes at 2 PDs (37 cells) and 59 PDs (283 cells). Bar, 200 µm. B, histogram showing side-by-side comparison of invasive activity (% of control) in early cultures ({square}) and late cultures ({blacksquare}). Results are shown for the CS-2 line (2 PDs versus 106 PDs), the CS-3 line (2 PDs versus 59 PDs), the CS-4 line (2 PDs versus 58 PDs), the CS-8 line (2 PDs versus 43 PDs), and a normal chondrocyte population (2 PDs versus 18 PDs). Columns, means based on nine determinations; bars, SDs. NC, normal chondrocyte population.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The data presented here support the hypothesis that telomerase expression in at least some human chondrosarcoma cells is associated with enhanced cell proliferation, morphological transition, and invasive behavior. Our analysis revealed strong telomerase activity in 5 of 16 human chondrosarcoma cell lines and weak activity 2 additional lines. Two of the positive cell lines (FS and JJ) had been cultured for several years before our investigation; thus, we cannot conclude that the original tumors from which they were derived would have shown similar levels of activity. However, high levels of telomerase activity were found in 3 tumor cell isolates at early passages (CS-6, CS-8, and CS-9), indicating that the original chondrosarcomas harbored significant numbers of telomerase-expressing cells. Telomerase activities in these cell populations were near the level expressed in a chondrocyte line transduced with hTERT. This activity was shown previously to maintain telomere length over >100 PDs in the hTERT-transduced line, suggesting that telomerase activity in the chondrosarcoma cell lines is also sufficient for long-term telomere maintenance. Neither enchondroma cell line showed telomerase activity, suggesting that these benign tumors lacked significant numbers of telomerase- expressing cells. The lack of telomerase activity in early- passaged chondrosarcoma cell cultures (CS-2, CS-3, CS-4, and CS-5) also indicated that there were few telomerase- expressing cells in the original tumors.

The FS, JJ, BG, and 105 lines had been cultured for an undetermined number of passages before we began the current study and grew for an additional 50 PDs at a steady rate in our laboratory. These findings indicate that the lines are effectively immortalized. Both the FS and JJ cell lines were strongly positive for telomerase activity, suggesting telomerase-dependent telomere maintenance. The lack of telomerase activity in the BG and 105 lines suggests these cells used the ALT mechanism of telomere maintenance. The same cell lines showed unusually heterogeneous telomere lengths on Southern blots, a finding consistent with the ALT mechanism (data not shown; Refs. 31 , 32 ).

Comparisons of initial telomere length showed that there was no significant difference in MTL between telomerase-positive and telomerase-negative cell lines. Rather, significantly longer telomeres were found among the recently isolated "CS" lines than among the long-term cultured FS, JJ, 105, and BG lines. These data indicated that extensive in vitro telomere erosion occurred before stabilization by telomerase activation (in the JJ and FS lines) or by the ALT mechanism (in the BG and 105 lines). It has been demonstrated that telomerase activation can occur as a postcrisis event and that telomerase can stabilize short telomeres without a causing a net gain in telomere length (40 , 41) . This implies that steady-state telomere length in telomerase-positive cells depends on telomere length at the onset of telomerase activation. Therefore, some heterogeneity in postcrisis telomere length is expected in different cell lines (42) .

Culture studies were undertaken with newly isolated CS lines to determine their long-term growth characteristics. We followed growth in 3 telomerase-negative lines (CS-2, CS-3, and CS-4) and 1 telomerase-positive cell line (CS-8). After growth to >60 PDs, it was apparent that initially telomerase-negative lines surpassed normal chondrocyte population growth barriers imposed by replicative senescence. This indicated that telomere maintenance mechanisms had been acquired at some point during the culture of the CS lines. Consistent with this hypothesis, we found that telomerase expression had increased from background levels to >1.5 RTA units. These findings showed that the extended growth of the cell lines was supported by the acquisition of telomerase activity. Telomerase expression in the CS-8 remained stable with time in culture.

Because suppression of one or more cell cycle control systems is a common finding in human malignancies, we compared p16INK4a, p21waf, pRb, and p53 levels in pre- and postplateau cells. Western blots showed that all these proteins were expressed in preplateau cells, but that p16INK4a was undetectable in postplateau cells. Alterations in the p16INK4a gene were reported previously in 9 of 22 human chondrosarcoma tissues. No mutations were found in the p53 gene (8 , 9) . All p16INK4a alterations were found in malignant, grade II or III tumors, although only 6 of 15 malignant tumors were affected. The majority of the alterations (4 of 6) were increases in methylation rather than deletions of the p16INK4a gene. Hypermethylation has been shown to silence transcription of the p16INK4a gene in a wide variety of human tumors (43) , suggesting that this is the mechanism leading to low p16INK4a protein levels in chondrosarcomas. Thus, our results agree with previous studies that strongly suggest that loss of p16INK4a expression is a key event in the malignant transformation of human chondrosarcoma cells. In addition, we found that the hyperphosphorylated (inactive) form of pRb increased with time, a phenomenon associated with loss of p16INK4a in many cell types. We also observed that p53 expression was increased dramatically in the late cultures, whereas p21waf was reduced slightly. Previous studies of the human chondrosarcoma indicate that p53 overexpression is associated with a variety of point mutations in the p53 gene, which disrupt its function (44) . These findings suggest that at least some of the overexpressed p53 observed in our CS cell lines is in a mutant, nonfunctional state that is incapable of activating p21waf transcription.

The p16INK4a-negative cells that emerged from the CS cultures after 5–10 PDs were morphologically distinct from their p16INK4a-expressing predecessors. The change from large, flattened, fibroblastic-appearing cells to smaller, more refractile cells indicates a reduction in substrate adhesion. A recent study of human pancreatic carcinoma cells revealed that, in addition to its function in regulating the cell cycle, p16INK4a modulates the expression of the {alpha}5 subunit of the {alpha}5ß1 integrin that serves as a fibronectin receptor (45) . The data showed that p16 INK4a reexpression in cells that had lost p16INK4a up-regulates expression of the {alpha}5 gene by 4–5-fold. These findings suggest that the more rounded cell shape and increased refractivity we observed in p16INK4a-negative CS cells was caused by reduced integrin expression.

Matrigel penetration assays were used to compare invasive activity in early- and late-passaged cultures. Activity levels reached near 40% in late-passaged cells from each line, a value comparable with published values for the HT1080 fibrosarcoma cell line (46) . This represented a striking, 4-fold increase relative to the early-passaged culture of the CS-2 line. Significant increases were also observed in the CS-3 and CS-4 lines, but these were less striking than for the CS-2 line. In contrast, there was no significant difference for the CS-8 line, which started out with a high level of activity and remained highly active at later PDs.

We initially expected that the telomerase-negative CS-2, CS-3, and CS-4 cell lines would eventually senesce because of replicative exhaustion of telomeres. The brief growth rate plateau we observed in all three lines coincided with rapid telomere erosion, suggesting that some cells in these populations did undergo replicative senescence. Senescence-induced G1 growth arrest is mediated by the p16INK4a/pRb tumor suppressor system (47 , 48) . Because both of these proteins were readily detectable in preplateau cells but p16INK4a was absent in postplateau cells, we conclude that loss of p16INK4a expression allowed some cells to overcome G1 arrest and proceed to telomere-induced crisis. This process has been shown to result in genomic instability and to the acquisition of a malignant phenotype (cell shape change, increased invasiveness, and telomerase activation; Refs. 49 , 50 ).

Our study of the CS-2, CS-3, and CS-4 cell lines showed that population growth rates increased with changes in morphology, loss of p16INK4a, and the acquisition of telomerase activity. One study of chondrosarcoma pathology specimens found a significant correlation between tumor grade and proliferation index (51) , suggesting that high growth rates are associated with malignant progression of the chondrosarcoma. These findings support the hypothesis that the changes we observed in vitro are related to malignant progression in vivo.

In contrast to the CS-2, CS-3, and CS-4 lines, the CS-8 line was initially positive for telomerase activity and maintained telomere length throughout in vitro growth. The subsequent in vitro behavior of this line suggested that many of the changes attributable to emergence from crisis had already taken place before the cells were isolated. Growth curves for this line did not show a distinct plateau phase, indicating that the majority of these cells did not experience replicative senescence after their isolation. Although we did observe progressive loss of p16 INK4a expression, increased p53 expression, and decreased p21waf expression, it is noteworthy that these changes were less dramatic than in the other cell lines. Similarly, invasion assays showed a high degree of activity in both early and late CS-8 cultures, indicating that invasive potential had already been acquired before the cells were placed in culture. These observations are consistent with the idea that that the majority of CS-8 cells had already undergone transformation before their isolation.

Our data do not support the conclusion that in vitro telomerase activity correlates with histological grade; cells from 50% (1 of 2) grade I tumors were positive for activity, and cells from 67% (4 of 6) grade III tumors did not show activity. Similarly, we found no striking relationship between telomerase activity and clinical outcome as measured by local recurrence or metastasis within 1 year of diagnosis as cells from 43% (3 of 7) "aggressive" tumors did not show activity and at least one cell line from a "nonaggressive" tumor did show activity. The latter finding must be interpreted with caution because malignant transformation of the chondrosarcoma can be delayed for years or decades (1) . Moreover, it is possible that a small fraction of tumor cells present at any single time point ultimately determine clinical outcome, or that malignancy is acquired as the tumor grows. Indeed, our long-term cell culture experiments suggest that cell populations from cartilage neoplasms are unstable and that aggressive cells can rapidly emerge from populations that appear relatively indolent.

Our study revealed a mechanism whereby cartilage neoplasms might be transformed from slowly growing cartilaginous masses that cause only local pressure effects on normal tissues to aggressive tumors that invade normal tissues and metastasize widely. These data show that telomerase activation, in concert with loss of p16INK4a expression, supports population growth well beyond the limits of normal chondrocytes. In addition the overexpression of p53 and underexpression of p21waf suggest that p53 mutation may also contribute to loss of growth control. Moreover, the data indicate that malignant characteristics are acquired as a consequence of passage through replicative crisis. Although a greater number of samples must be analyzed before firm conclusions can be drawn, our current findings support the intriguing possibility that telomerase expression and the absence of p16INK4a could serve as a prognostic markers of the potential for aggressiveness, as has been suggested for lung cancer (52) . This strategy may help identify apparently benign or histologically low-grade cartilage neoplasms that have a high risk of malignant behavior.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Human chondrosarcoma cell lines 105KC (10 , 11 , 36 , 37) , 4BG, FS, and JJ (11) were thawed from frozen stocks. Additional human chondrosarcoma cell lines (CS-2, CS-3, CS-4, CS-5, CS-6, CS-7, CS-8, CS-9, CS-10, CS-11, CS-12, and CS-13) and enchondroma lines (EN-1 and EN-2) were freshly established from tumors. A normal cell strain was established from normal tibial plateau cartilage isolated from a 47-year-old donor. Chondrosarcoma cells, enchondroma cells, and normal chondrocytes were isolated by overnight digestion of tissues in growth medium containing 0.5 mg/ml type IA collagenase and Pronase E (Sigma Chemical Co.). A telomerase-expressing chondrocyte cell line was used as positive control (38) . All cells were cultured as monolayers in growth medium containing 40% DMEM, 40% MEM, 20% Ham’s F-12, and 10% FCS (Life Technologies, Inc.) supplemented with 1.0 units/ml insulin, 20 µg/ml hydrocortisone (Sigma), and 40 µg/ml lentamicin or 100 units/ml penicillin/streptomycin. Phase-contrast micrographs of the cultures were taken using a x20 objective on an Olympus inverted microscope and T-Max 100 film (Kodak).

Cells freshly isolated from tumors (CS-2 through CS-13) were counted manually, and primary cultures were established in T-75 flasks (Costar). Frozen cell stocks (JJ, FS, BG, and 105) were thawed, counted, and inoculated in T-75 or T-225 flasks. The cells were grown until confluent and passaged using trypsin/EDTA. A portion of the primary cultures (or initial frozen cell inoculates) were stored for telomerase activity assays. The JJ, BG, FS, 105, CS-2, CS-3, and CS-4 lines were re-inoculated in 6-well tissue culture plates (50,000 cells/well). These secondary cultures were incubated until confluent (4–15 days) with regular medium changes (every 2–3 days), then trypsinized, counted, and re-inoculated in fresh 6-well plates. This process was repeated for up to 6 months. Cumulative PDs were calculated from cell counts at each passage. Excess cells at each passage were stored under liquid N2 in culture medium containing 10% DMSO or were washed in PBS and frozen at -80°C for DNA analysis.

Telomerase activity was determined using a TRAP assay kit (Roche, Indianapolis, IN) according to the manufacturer’s instructions. In this protocol, telomerase activity is measured by ELISA of products generated by telomerase elongation of an artificial telomeric repeat sequence primer. These elongated products are subsequently amplified by PCR and quantified. Products containing the biotinylated primers used as templates in the elongation reaction are immobilized on streptavidin-coated 96-well plates and probed with telomeric repeat DIG-labeled oligonucelotides. The DIG label is detected using an anti-DIG antibody conjugated to horseradish peroxidase with tetramethylbenzidine as a substrate. The absorbance (OD) at 450 nm was measured on a multiwell plate reader (Molecular Devices, Sunnyvale, CA). The version of the assay we used allowed relative quantitation of telomerase activity, provided extracts are prepared from equal numbers of cells. Accordingly, we used a standard number of cells (1 x 106) and volume of extract buffer (0.5 ml) for each determination. In addition, the following suggested controls were incorporated: an internal control present in all samples which consisted of an amplifiable telomere repeat sequence of known concentration; negative controls consisting of heat-inactivated samples or extract buffer; and a positive ("low" positive) control extract from a telomerase-positive cell line. RTA of the samples was calculated as follows: [(ODsample - ODheat-inactivated sample)/ODinternal standard]/[(ODpositive control extract - ODextract buffer)/ODinternal standard]. TRAP assay PCR products were analyzed by PAGE essentially as described in the TRAP assay kit. Briefly, 20 µl of each reaction were fractionated on 12% nondenaturing gels and blotted onto positively charged nylon membranes by electrotransfer in Tris-borate buffer. The blots were probed using a Biotin Luminescence Detection kit (Roche).

Genomic DNA was isolated from ~1 x 106 to 5 x 106 cells using a DNeasy kit (Qiagen) according to the manufacturer’s directions. The DNA concentration of each sample was determined by UV spectrophotometry, and 2 µg were digested to completion with 10 units each of RsaI and HinfI (New England Biolabs) in a 60-µl reaction. The reactions were electrophoresed on 0.8% SeaKem Gold agarose (FMC Bioproducts) in parallel with DIG-labeled {lambda} HindIII size standards (Roche). The gels were transferred by capillary action to Hybond-N+ (Amersham) nylon membranes in x20 SSC and irradiated with 1200 µJ of UV light. Nonradioactive methods were used to detect telomere sequences according to Genius system directions published by the manufacturer (Roche). In brief, the membranes were prehybridized for 4–16 h at 37°C in hybridization buffer (50% formamide, 5x SSC, 0.1% sodium lauryl sulfate, 0.02% SDS, and 2% block). A synthetic oligonucleotide complimentary to human telomeric repeat sequences [(CCCTAA)3] and labeled at the 3' end with DIG (Sigma; Genosys) was diluted to 50 pM in hybridization buffer, and the membrane was probed for 16–24 h at 37°C. Excess probe was removed by washing the membranes twice in 2x SSC with 0.1% SDS at ambient temperature (2 x 15 min) and then in 0.5x SSC with 0.1 SDS at 37°C (2 x 15 min). A goat anti-DIG alkaline phosphatase-conjugated antibody and a chemiluminescent substrate, CDP-Star (Roche), were used to detect the DIG-labeled probe. Autoradiograms of the blots (15–120-min exposures) were digitized using a flat bed scanner (Hewlett Packard ScanJet II CX). Absorbance scans of each lane were performed using Scion Image (Scion Corp.) on a personal computer. The positions of the {lambda} HindIII standard bands were plotted (log molecular weight versus relative migration distance), and the data were fitted using linear regression analysis (Microcal Origin). Mean telomere terminal restriction fragment lengths (MTLs) were derived as described (53 , 54) .

Western blot analyses were performed using extracts from subconfluent cultures at early (<5 PD) and late (>40 PD) time points. Cells were extracted in lysis buffer [25 mM Tris-HCl (pH 7.5), 125 mM NaCl, 2.5 mM EDTA, 0.05% SDS, 0.5% NP40, 0.5% deoxycholate, and 10 glycerol] containing protease and phosphatase inhibitors (1.0 mM phenylmethylsulfonyl fluoride, 25 µg/ml leupeptin, 10 µg/ml pepstatin, 1.0 µg/ml aprotinin, 100 mM ß-glycerol phosphate, 0.5 mM sodium vanadate, and 50 mM sodium fluoride). Total protein concentration was determined by bicinchoninic acid assay using a commercial kit (Pierce, Rockford, IL). Ten µg of total protein were loaded in all lanes except for the p16INK4a late-passaged lanes, which were loaded with 20 µg of total protein to ensure detection of lower levels of tumor suppressor proteins. The samples were fractionated on 8% (pRb and p53 analysis) or 16% (p16INK4a and p21waf1 analysis) reducing SDS-polyacrylamide gels. Fractionated proteins were electroblotted onto Immobilon-P nylon membranes (Millipore). The blots were probed by overnight incubation (4°C) with one of the following primary antibodies: mouse monoclonal antihuman pRb AB-11 (Oncogene Research Products) diluted to 1.0 µg/ml; mouse monoclonal antihuman p21waf1 Ab-1 (Oncogene Research Products, Boston, MA) diluted to 0.5 µg/ml; mouse monoclonal antihuman p53 Ab-6 (Calbiochem, San Diego, CA) diluted to 1.0 µg/ml; and mouse monoclonal anti-human p16INK4a (BD PharMingen, San Diego, CA) diluted to 1.0 µg/ml. The probed blots were washed extensively and probed with a goat-antimouse alkaline phosphatase-conjugated secondary antibody (Promega Corp., Madison, WI) diluted 1:15,000. Blots were washed extensively, and target proteins were detected using CDP-Star (Roche) and Biomax MR autoradiography film (Eastman Kodak, Rochester, NY).

Invasion assays were performed in BioCoat Matrigel invasion chambers essentially as described by the manufacturer (BD Biosciences). Chondrosarcoma cells and normal chondrocytes (25,000–50,000) were suspended in 0.5 ml of modified culture medium (standard growth medium without insulin and with 1.0% FCS) were seeded in invasion chambers (previously rehydrated for 2 h in the same medium). The invasion chambers were placed in 24-well plates over 0.75 ml of standard growth medium, and the plates were incubated for 24 h at 37°C. Controls consisting of chambers without Matrigel coating were included in each assay. After 24 h, noninvading cells were removed from the top surface of the chambers, and the membranes were fixed in 100% methanol and stained with toluidine blue. The stained membranes were dried and mounted in immersion oil on microscope slides. The mounted membranes were photographed using transmitted light optics on an Olympus BX 51 microscope equipped with a digital camera. Cells per x10 field were counted manually. Invasion percentage was scored by dividing the number of cells in the invasion chamber by the number in the control chamber (x100). Each assay was performed in triplicate chambers, and three fields/chamber were used for cell counts.

Statistical Analysis.
Differences between telomere length, telomerase expression, and invasion percentage were tested for statistical significance using Student’s t test.


    Acknowledgments
 
We are grateful to Terese Nickol for expert technical assistance.


    Footnotes
 
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.

1 This work was supported by the Ben Ling Chondrosarcoma Research Fund and the University of Iowa Department of Orthopaedic Surgery. Back

2 To whom requests for reprints should be addressed, at Department of Orthopedic Surgery, Biochemistry Lab, The University of Iowa, 1182 ML, Iowa City, IA 52242. Phone: (319) 335-7550; Fax: (319) 335-6879; E-mail: james-martin{at}uiowa.edu Back

3 The abbreviations used are: hTERT, human telomerase reverse transcriptase; RTA, relative telomerase activity; MTL, mean telomere length; PD, population doubling; TRAP, telomere repeat amplification protocol; DIG, digoxigenin. Back

Received for publication 3/26/02. Revision received 6/21/02. Accepted for publication 6/24/02.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
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