Affiliations of authors: J. Albanell, M. Engelhardt, M. A. S. Moore, Laboratory of Developmental Hematopoiesis, Cell Biology Program, Sloan-Kettering Institute, New York, NY; G. J. Bosl (Department of Medicine), V. E. Reuter (Department of Pathology), Memorial Hospital, Memorial Sloan-Kettering Cancer Center, New York, NY; S. Franco, Laboratory of Developmental Hematopoiesis, Cell Biology Program, Sloan-Kettering Institute, and Department of Pediatrics, Memorial Hospital, Memorial Sloan-Kettering Cancer Center; E. Dmitrovsky, Laboratory of Molecular Medicine, Molecular Pharmacology and Therapeutics Program, Sloan-Kettering Institute, and Department of Medicine, Memorial Hospital, Memorial Sloan-Kettering Cancer Center.
Correspondence to: Ethan Dmitrovsky, M.D., Remsen 7650, Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH 03755-3835 (e-mail: ethan.dmitrovsky{at} dartmouth.edu).
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ABSTRACT |
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INTRODUCTION |
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Germ cell tumors are unique in their capacity to undergo extensive differentiation, known as teratoma formation (10). Unlike undifferentiated embryonal carcinomas from which teratomas derive, mature teratomas have limited proliferative capacity, although malignant transformation can occur (11). This biologic feature provides an opportunity to investigate in the clinical setting how telomerase activity and tumor cell differentiation state relate. Studies investigating telomerase activity in male germ cell tumors are also relevant because these tumors derive from germ cell progenitors that constitutively express telomerase to regulate their telomere lengths (2,3,6,12). These findings are of added interest because late-generation telomerase RNA null mice exhibit defective spermatogenesis with increased apoptosis and decreased proliferation rates observed in the testes (13). This suggests that regulation of telomerase activity and perhaps telomere lengths are required for the maturation of normal or transformed germ cells.
Male germ cell tumors are broadly classified as seminomas and nonseminomas. These tumors exhibit diverse histopathology, often with extensive somatic differentiation (14). This study was undertaken to contrast telomerase activities and telomere lengths in male germ cell cancers (seminomas, nonseminomas, and mixed germ cell tumors) with those in mature or immature teratomas to understand the relationship between the telomerase activity and the differentiation state of clinical germ cell tumors. A modified telomeric repeat amplification protocol (TRAP) assay (6,15,16) was used to quantify telomerase activities.
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MATERIALS AND METHODS |
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Forty-one tumor specimens were obtained from 35 patients having germ cell tumors, who underwent potentially curative or diagnostic surgical resections. Benign testicular tissues were obtained either from patients who underwent orchiectomy for prostate cancer or from patients who underwent orchiectomy for germ cell cancer and had adjacent benign testicular tissue available for examination. Use of these found tissue specimens was approved by the Institutional Review Board. Within 10 minutes of surgical resection, the specimens obtained were snap-frozen in liquid nitrogen. Histopathologic analyses confirmed that extensive lymphocytic infiltrates and germinal centers or contaminating normal tissues were not present.
Germ cell tumors often exhibit histopathologic heterogeneity. To confirm the histopathologic diagnosis of the specimens used for telomerase and telomere measurements, a portion of each frozen tissue specimen was also sent for histopathologic analyses. All tissue specimens were reviewed by a single reference pathologist (V. E. Reuter) to confirm the histopathology present and to exclude concurrent pathologic processes. For those specimens used for TRAP assays, terminal restriction fragment (TRF) length and alkaline phosphatase measurements were immediately adjacent to those processed for histopathologic diagnoses. This permitted statistical correlations to be made between telomerase activities, telomere lengths, and the histopathology of the examined germ cell tumors. A portion of the same specimen was available for protein extraction and for isolation of genomic DNA used to assess TRF lengths.
Protein Extraction
Frozen tissue specimens (50-100 mg) were homogenized in 100-200 µL of ice-cold CHAPS (3-[{3-cholamidopropyl}-dimethylammonio]-1-propane-sulfonate) lysis buffer by use of disposable pestles, incubated on ice for 30 minutes, and centrifuged at 12 000g for 30 minutes at 4 °C. The supernatant was immediately collected, and the protein concentration was measured by use of the BioRad protein assay kit (Bio-Rad Laboratories, Richmond, CA). Protein aliquots were stored at -80 °C as 1 µg/µL stocks, as previously described (6,8). The isolated protein extracts were independently analyzed for alkaline phosphatase activities, as previously reported (16). This analysis was used to confirm that protein extracts used for TRAP assays were of sufficient integrity to measure another enzymatic activity susceptible to degradation in clinical tissues. If alkaline phosphatase activities were not detected in isolated protein extracts, then those extracts were not used for subsequent TRAP analyses.
TRAP Assay
The telomerase TRAP assay was performed by use of a modified polymerase chain reaction (PCR)-based method, previously established (15,16). Two micrograms of desired protein extracts was assayed in reactions containing 50 µL of the TRAP reaction mixture. For each assay, a negative control and 0.1 amol of the quantitation standard oligonucleotide R8 were included. The results were quantitated as previously reported (16). Briefly, this assay incorporates an internal PCR control of a 36 base-pair (bp) product (designated TSNT), running 14 bp below the smallest size-fractionated, TRAP-derived species. The amount of telomerase activity (total product generated [TPG]) for each reaction was calculated by use of the formula:
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T = radioactive counts from telomerase bands from the protein extracts, B = radioactive counts from a negative control (background), R8 = radioactive counts from R8 (0.1 amol), CT = radioactive counts from the internal control TSNT (0.01 amol) of the protein extract, and CR8 = radioactive counts from TSNT (0.01 amol) of the R8 (0.1 amol). One unit of TPG was defined as 0.001 amol or 600 molecules of telomerase substrate (TS) primer (15) extended by at least three telomeric repeats by the telomerase activity present in the examined extract and corresponds approximately to the activity present within a single immortal cell. Linearity of the assay was confirmed over at least three logs of the target protein concentrations [(16); data not shown]. All of the protein extracts were analyzed in at least two independent TRAP assays. The average telomerase activity (TPG) was calculated for every analyzed specimen. Presence of a potential telomerase inhibitor in telomerase-negative specimens was assayed by TRAP experiments by use of a protein extract from a mature teratoma mixed with protein extract from a neuroblastoma cell line known to have telomerase activity without a telomerase inhibitor.
TRF Length Measurements
TRF length measurements were performed by use of 10 µg of genomic DNA digested with the restriction endonucleases MspI and RsaI (Boehringer Mannheim GmbH, Mannheim, Germany) and electrophoresis on a 0.5% agarose gel. Hybridizations with telomere-specific (TTAGGG)3 radiolabeled oligonucleotide probe were performed. Mean TRF lengths were calculated, as previously reported (16-18).
Statistical Analyses
When several assays were performed on the same tissue specimen or several tissue specimens from the same patient were analyzed, the average results from these assays were used for statistical analyses. Mean and standard deviation (SD) values for TPG and TRF length measurements were assessed for each histopathologic subtype. The association between telomerase activity in mature teratoma compared with that of germ cell cancers was examined by Fisher's exact test. Fisher's exact test was calculated by use of BMDP version 1.1 for Windows. All P values are two-sided. A two-sided Pearson's correlation test was used to analyze the correlation between telomerase activities and telomere lengths. Differences between mean telomerase activity and telomere lengths by histopathologic subtype were analyzed by the one-way ANOVA (analysis of variance) test.
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RESULTS |
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The absence of telomerase activity in teratomas was in marked contrast to the high levels
detected in the examined germ cell cancers. Notably, a single immature teratoma having
malignant
transformation had high telomerase activity (data not shown). This provides independent
confirmation of a link between telomerase activity present in teratomas and the malignant
potential
of these tumors. Very long telomeres were detected in some mature teratomas (see Fig.
2), indicating that telomerase repression can represent a late event in
teratoma formation. To confirm that the telomerase activity measured in seminomas was not due
to an extensive lymphoid infiltrate that may include germinal centers the reference pathologist
(V. E. Reuter) reviewed the histopathology of these cases. These dissected tumors were not
found to have extensive lymphoid infiltrates or germinal centers (data not shown).
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DISCUSSION |
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Some human tumors have no detectable telomerase activity (20).
Several mechanisms may contribute to this finding. Telomerase reactivation may not be required
when tumor precursor cells have long telomere lengths and transformation results from few
mutations. This is suggested by the absence of telomerase activity found in some retinoblastomas
(21). The absence of telomerase activity in some advanced stage
neuroblastomas (22) may reflect tumor cell maturation. It is this
mechanism for telomerase repression that is proposed as active in mature teratomas. It is notable
that long telomeres were detected in some mature teratomas, indicating that telomerase
repression
is not an early step in teratoma formation. TRF lengths greater than or equal to 20 kb were
reported in sperm and fetal tissues (2,3). Three germ cell tumors in this
study were found to have long telomeres and high telomerase activity. In other examined germ
cell
tumors, short TRF lengths (defined as TRF <10 kb) were measured with detectable telomerase
activity, as shown in Table 2. However, the mean TRF lengths in the
different germ cell tumor types were compared with a one-way ANOVA analysis and were not
found to demonstrate a statistically significant difference (P = .069).
Telomerase repression might result from tumor cells exiting the cell cycle. An association is previously reported between telomerase activity and proliferation. Telomerase is repressed when tumor cells achieve quiescence (23-26). In quiescent hematopoietic progenitors, basal telomerase activity is low but is rapidly induced when cells enter the cell cycle after exposure to hematopoietic growth factors (25). Telomerase activity is inducible in some cultured somatic cells (26). A relationship between telomerase activity and proliferation is reported. In breast cancer, telomerase activity is associated with S-phase fraction in lymph node-positive cancers (27). In primary lung cancers, expression of the proliferation marker Ki-67 was associated with telomerase activity (16). In germ cell tumors, expression of Ki-67 and proliferating cell nuclear antigen were detected in almost all of the examined germ cell tumors, including mature teratomas (28).
Telomerase activity is regulated in spermatogenesis and in early embryogenesis (12,19). Telomerase activity is not detected in mature spermatozoa and unfertilized
eggs but is present in blastocysts and many fetal tissues. This developmental regulation of
telomerase may account for detection of different telomerase activities or TRF lengths in the
subsets of germ cell tumors present in Table 2. Male germ cell tumors are
among the most sensitive to chemotherapy, and advanced stage germ cell tumors are often cured
with cisplatin-based chemotherapy (14). Perhaps chemotherapy
treatments
will affect telomerase activities in germ cell tumors. It is not yet known whether chemotherapy
treatments alter telomerase activities in teratomas.
Alternative mechanisms may compensate for the end-replication problem and eliminate the need for telomerase activation in tumors. An alternative mechanism for the lengthening of telomeres was found in immortalized cell lines and in subsets of tumor-derived lines (29). Some mature teratomas in this study had long telomeres. This is reminiscent of the long telomeres detected when alternative mechanisms for telomere lengthening are present (29). However, telomere lengths similar to or shorter than those found in the benign testicular tissues (TRF length 14.3 kb; range, 12.52-14.4 kb) were observed in other teratomas. Therefore, an alternative mechanism is not likely to provide a consistent explanation for telomere lengthening in these mature teratomas. Other reasons for the absence of telomerase activity in teratomas may exist. Although an inhibitor of telomerase activity was not found, telomerase activation may precede repression after telomere lengthening in teratomas. Telomerase activity in teratomas may occur at levels below detection by this TRAP assay. While these or alternative telomere lengthening mechanisms are not formally excluded, the absence of telomerase activity in mature teratomas is hypothesized to result from signaling of tumor cell differentiation.
In summary, this study reports that telomerase activity was detected in all of the examined germ cell cancer specimens. In marked contrast, telomerase activity was not detected in the examined mature teratomas. No telomerase inhibitory activity was detected in any of these teratomas, and the integrity of the protein extracts used in this study was confirmed. These findings indicate that an inverse relationship exists between telomerase activity and the differentiation state of germ cell tumors. Notably, an immature teratoma exhibiting malignant transformation had telomerase activity detected. Thus, telomerase activation is commonly found in malignant germ cell tumors without extensive evidence of maturation. Absence of telomerase activity is a consistent feature of mature teratomas. Since long telomeres were detected in some mature teratomas, telomerase repression can be a late event in teratoma formation. Taken together, this study offers evidence for a direct link between telomerase activation and differentiation state of clinical germ cell tumors. Future work will determine whether this absence of telomerase activity is a marker or cause of teratoma formation.
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NOTES |
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Present address: M. Engelhardt, Hematology/Oncology Department, University of Freiburg, Germany.
Present address: E. Dmitrovsky, Departments of Medicine and Pharmacology and Toxicology, Norris Cotton Cancer Center, Dartmouth Medical School, Hanover, NH.
Supported by American Cancer Society grant RPG-90-019-08-DDC (E. Dmitrovsky); by Public Health Service grants R01CA54494-06 (E. Dmitrovsky) and U19CA67842-02 (M. A. S. Moore) from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services; by the Gar Reichman Fund of the Cancer Research Institute (M. A. S. Moore); by Fondo de Investigacion Sanitaria (Spain) grant 96/5706 (J. Albanell); by Comissio Interdepartmenta per a la Recerci i Innovacio Technologica (Spain) grant 1996 BEAI200087 (J. Albanell); and by grant Deutsche Forschungsgemeinschaft (Germany) 95/3191/1-01 (M. Engelhardt). M. A. S. Moore conducts research sponsored by Geron Corporation, Menlo Park, CA.
We thank Dr. N. W. Kim, Geron Corporation, Menlo Park, CA, for providing the modified Telomeric Repeat Amplification Protocol assay methodology used in this study. We also thank Ms. Geralyn Higgins for her assistance in the review of the clinical features of the patients from whom the germ cell tumors were derived and Dr. Silvia Sauleda, Hospital Universitari Vall d'Hebron, for her helpful consultation regarding statistical analyses.
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Manuscript received December 18, 1998; revised June 1, 1999; accepted June 7, 1999.
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