Antisense telomerase treatment: induction of two distinct pathways, apoptosis and differentiation

^a Department of Neurosurgery, Brain Tumor/Neuro-Oncology Center, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA
^b Department of Neurosciences, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA
^c Department of Molecular Biology, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA
^d Department of Microbiology, The Tokyo Metropolitan Institute of Medical Science, Tokyo 113, Japan
^e Department of Hematology, Research Institute, International Medical Center of Japan, Tokyo 162, Japan
^f Geron Corporation, 200 Constitution Drive, Menlo Park, California 94025, USA

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Telomerase, the enzyme that elongates telomeric DNA (TTAGGG)_n, may be involved in cellular immortality and oncogenesis. To investigate the effect of inhibition of telomerase on tumor cells, we transfected the antisense vector against the human telomerase RNA into human malignant glioma cells exhibiting telomerase activity. After 30 doublings, some subpopulations of transfectants expressed a high level of interleukin-1ß-converting enzyme (ICE)² protein and underwent apoptosis. In contrast, other subpopulations also showed enhanced ICE protein but escaped from apoptotic crisis and continued to grow, although their DNA synthesis, invasive ability, and tumorigenicity in nude mice were significantly reduced. Surviving cells demonstrated increased expression of glial fibrillary acidic protein and decreased motility, consistent with a more differentiated state. These cells also contained enhanced expression of the cyclin-dependent kinase inhibitors (CDKIs) p21 and p27. Treatment of surviving nonapoptotic cells with antisense oligonucleotides against p27, but not p21, induced apoptotic cell death, suggesting that p27 may have protected differentiating glioma cells from apoptosis. These data show that treatment with antisense telomerase inhibits telomerase activity and subsequently induces either apoptosis or differentiation. Regulation of these two distinct pathways may be dependent on the expression of ICE or CDKIs.—Kondo, S., Tanaka, Y., Kondo, Y., Hitomi, M., Barnett, G. H., Ishizaka, Y., Liu, J., Haqqi, T., Nishiyama, A., Villeponteau, B., Cowell, J. K., Barna, B. P. Antisense telomerase treatment: induction of two distinct pathways, apoptosis and differentiation FASEB J. 12, 801–811 (1998)

Key Words: ICE • p27 • CDKI • tumor cells • tumorigenicity • oglionucleotide

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
MALIGNANT GLIOMAS are the most common malignant brain tumors and are considered to be incurable (1). The activation of certain proto-oncogenes and inactivation of tumor suppressor genes are considered to be key mechanisms for the development and progress of tumors (2–4). The precise molecular events, however, are unknown. In the process of cellular oncogenesis, separate or complementary mechanisms may also play an important role (5).

Telomeres correspond to the ends of eukaryotic chromosomes and are specialized structures containing unique (TTAGGG)_n repeats (6). Telomeres protect the chromosomes from DNA degradation, end-to-end fusions, rearrangements, and chromosome loss (7). Because cellular DNA polymerases cannot replicate the 5' end of the linear DNA molecule, the number of telomere repeats decreases (by 50–200 nucleotides/cell division) during aging of normal somatic cells (8, 9). Shortening of telomeres may control the proliferative capacity of normal cells. Telomerase, a ribonucleic acid–protein complex, adds hexameric repeats of 5'-TTAGGG-3' to the end of telomeres to compensate for the progressive loss (10). Although normal somatic cells do not express telomerase, immortalized cells such as tumors express this activity (11–16). More recently, HeLa cells transfected with an antisense human telomerase were found to lose telomeric DNA and to die after 23 to 26 doublings (17). However, the precise molecular mechanisms remain unclear. In this study, we determined the effect of antisense against the human telomerase RNA on human malignant glioma U251-MG cells with telomerase activity. We report here that inhibition of telomerase activity directs malignant glioma cells to two distinct pathways: apoptosis and differentiation. The enhanced expression of interleukin-1ß-converting enzyme (ICE),² a mammalian homolog of the Caenorhabditis elegans cell death gene ced-3 (18) that has been identified as the regulator of apoptosis in several cell types (19–21), was associated with both pathways. Enhanced expression of the cyclin-dependent kinase inhibitors (CDKIs), p21 and p27 (22–24), was associated with the differentiation pathway. These data suggest that ICE and CDKIs may be involved in the regulation of pathways to apoptosis and differentiation after shutdown of telomerase.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Tumor cells
Human glioblastoma U251-MG cells (25) were used in this study. Tumor cells were cultured in Dulbecco's modified Eagle's medium (DMEM, GIBCO BRL, Grand Island, N.Y.) supplemented with 10% heat-inactivated fetal calf serum (GIBCO BRL), 4 mM glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin.

Telomerase assay
The telomerase assay was performed by a previously described method (12) with some minor modifications (26). Tumor cells were seeded at 10⁴ cells/well (0.1 ml) in a 96-well microplate (Corning, Corning, N.Y.) and incubated overnight at 37°C. Cells were washed once in phosphate-buffered saline and homogenized in 50 µl of ice-cold lysis buffer [10 mM Tris-HCl (pH 7.5), 1 mM MgCl₂, 1 mM EGTA, 0.1 mM PMSF, 5 mM ß-mercaptoethanol, 0.5% CHAPS, 10% glycerol]. After 30 min of incubation on ice, the lysates were centrifuged at 100,000 g for 30 min at 4°C, and the supernatant was rapidly frozen and stored at -80°C. The concentration of protein was measured with the use of the BioRad Protein Assay (Richmond, Calif.); an aliquot of extract containing 5 µg/µl of protein was used for each telomerase assay. For RNase treatment, 5 µl of extract was incubated with 1 µl RNase A (1 mg/ml) for 30 min at 25°C. Two microliters of each extract were assayed in 50 µl of reaction mixture containing 20 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂, 63 mM KCl, 0.05% Tween-20, 1 mM EGTA, 50 µM deoxynucleoside triphosphates, 0.2 to 0.4 µl of [-³²P]dCTP (10 µCi/µl, 3000 Ci/mM), 0.1 µg of TS oligonucleotides (5'-AATCCGTCGAGCAGAGTT-3'), 1 µg of T4 gene 32 protein (Boehringer-Mannheim Biochemicals, Indianapolis, Ind.), and two units of Taq DNA polymerase (Boehringer-Mannheim). After a 10 min incubation at room temperature for telomerase-mediated extension of the TS primer, 0.1 µg of CX oligonucleotides (5'-CCCTTACCCTTACCCTTACCCTAA-3') was added. The reaction mixture was then subjected to polymerase chain reaction (PCR) amplification in a thermal cycler with 30 cycles at 94°C for 30 s, 50°C for 30 s, and 72°C for 1.5 min in the presence of an internal TRAP assay standard (ITAS, 36 bp; Oncor, Gaithersburg, Md.). The PCR product was electrophoresed in 0.5x Tris-borate EDTA on a 10% polyacrylamide gel. The gels were dried and autoradiography was performed for 24 h at -70°C. Radioactivity was also quantitated with a Molecular Dynamics PhosphorImager (Sunnyvale, Calif.).

Antisense human telomerase expression vector
The plasmid (p-10–3-hTR) containing cDNA of the antisense of human telomerase (17) was digested with EcoRI, and a 200 bp EcoRI DNA fragment (1 to 185 nucleotides of antisense telomerase) was purified as described previously (26). The fragment was inserted into the EcoRI site of the plasmid pBabe-puro (pBabe anti-telo). U251-MG cells were plated at a density of 5x10⁵ cells/ml 1 day before transfection of pBabe anti-telo vector. Cells were transfected with the pBabe anti-telo plasmid or control vector pBabe-puro by using lipofectamine-mediated gene transfer, as described previously (20). Selection with puromycin was started 48 h after transfection. To determine whether stable clones with the pBabe anti-telo had decreased telomerase activity, a telomerase assay was performed as described above.

Apoptotic features
To determine whether tumor cells transfected with the pBabe anti-telo plasmid displayed an apoptotic morphology, tumor cells were stained with the DNA binding fluorochrome bis (benzimide) trihydro-chloride, Hoechst 33258 (8 µg/ml), as described previously (27). Five hundred cells were counted and scored for the incidence of apoptotic chromatin changes (blebbing, fragmentation, and condensation) under UV-fluorescence microscopy. A DNA fragmentation assay in agarose gel was performed using methods previously described (28). The cells (1x10⁷) were lysed in 1.0 ml of a buffer consisting of 10 mM Tris-HCl, 10 mM EDTA, and 0.2% Triton X-100 (pH 7.5). After 10 min on ice, the lysate was centrifuged (13,000 g) for 10 min at 4°C in an Eppendorf microtube. The supernatant (containing RNA and fragmented DNA but not intact chromatin) was extracted first with phenol and then with phenol:chloroform:isoamyl alcohol (25:24:1). The aqueous phase was made to 300 mM NaCl; nucleic acids were precipitated with 2 vol of ethanol and then dissolved in 20 µl of 10 mM Tris-HCl-1 mM EDTA (pH 7.5). After digestion of RNA with RNase A (0.6 mg/ml, at 37°C for 30 min), the sample was electrophoresed in a 1.5% agarose gel with TAE buffer (40 mM Tris-acetate and 1 mM EDTA, pH 8.3). DNA was then visualized with ethidium bromide staining.

Immunohistochemical staining
For histopathological studies, tumor cells were seeded at 10⁵ cells/well (1 ml) in 4-well Lab-Tek chamber slides (Nalge Nunc International, Naperville, Ill.) and incubated overnight at 37°C. The cells were fixed with 1% formaldehyde and 0.2% glutaraldehyde for 5 min, rinsed three times with phosphate-buffered saline, and immunohistochemically stained using antibody to ICE (Santa Cruz Biotechnology, Santa Cruz, Calif.) (29).

Immunoblotting assay
Soluble protein for immunoblotting was harvested from tumor cells lysed in 500 µl of freshly prepared extraction buffer (10 mM Tris-HCl pH 7, 140 mM sodium chloride, 3 mM magnesium chloride, 0.5% NP-40, 2 mM phenylmethylsulfonyl fluoride, 1% aprotinin, 5 mM dithiothreitol) for 20 min on ice (27). Equal amounts of protein estimated by the BioRad Protein Assay were separated by electrophoresis on a 10% polyacrylamide gel in sodium dodecyl sulfate and thereafter subjected to electrotransfer to nitrocellulose. Filters were subjected to immunoblotting using the ECL (Amersham, Arlington Heights, Ill.) detection system according to the manufacturer's instructions. The specific antibodies used were glial fibrillary acidic protein (GFAP) (DAKO, Carpinteria, Calif.), ICE, p16, p27 (Santa Cruz Biotechnology), and p21 (Oncogene Science, Uniondale, N.Y.). Equivalent sample loading of the intact protein was confirmed by blotting, using the anti-actin (Boehringer-Mannheim) antibody. The intensity of each band was quantitated by densitometry.

DNA synthesis and cell cycle analysis
Determination of DNA synthesis was assayed by using the incorporation of [³H]thymidine (30). Tumor cells were seeded at 10⁴ cells/well (0.1 ml) in 96-well flat-bottomed plates (Corning) and incubated overnight at 37°C. The cells were pulsed with 5 µCi/ml of [³H]thymidine (Amersham) for 2 h and radioactive content was assayed in a liquid scintillation counter. Results were expressed as mean counts per minute ± SD. For cell cycle analysis, trypsinized tumor cells were stained with propidium iodide by using the Cycletest kit (Becton Dickinson, Mountain View, Calif.) and analyzed for DNA content by using the FACScan (Becton Dickinson), as previously described (31). Cell cycle distribution was determined using CellFIT Software (HP340 Series 9000 Workstation). Dead cells were gated out by using pulse processing.

Three-dimensional anchorage-independent cultures
To evaluate the growth and invasive properties of tumor cells transfected with the pBabe anti-telo, suspensions of 2x10⁴ cells in 50 µl of serum-free medium were mixed with 150 µl of Matrigel (Collaborative Research, Inc., Bedford, Mass.) and plated in 48-well plates (Corning) as described previously (29). The gel was allowed to solidify for 30 min at 37°C, after which 800 µl of DMEM containing 10% fetal calf serum was layered on top. Each well was monitored daily for morphological changes. After 3 and 7 days of incubation, each well was photographed by using an inverted photomicroscope.

Tumorigenicity assays
For assays of tumorigenicity, 2.5 x 10⁶ cells suspended in 0.1 ml serum-free DMEM and 0.1 ml Matrigel were injected subcutaneously into the flanks of 8- to 12-wk-old female Balb/cByJ athymic nude mice, as described previously (29). Animals were monitored regularly for tumor occurrence and size. All animal procedures were approved by the CCF Research Programs Committee.

Motility assay
To determine the effect of inhibited telomerase activity on motility of U251-MG cells, a motility assay was performed as described previously, with modifications (32). Briefly, tumor cells were seeded at 10⁵ cells/ml on plastic coverslips placed in 35 mm tissue culture wells (Corning) and incubated overnight at 37°C. For time lapse videomicrography, a 35 mm culture dish was maintained at 37°C in a 95% air/5% CO₂ environment in a stage heater (20/20 Technology, Whitehouse Stations, N.J.) mounted on a phase-contrast microscopy. A digital image was recorded every 5 min for 24 h using a charge-coupled device camera (Sierra Scientific, Sunnyvale, Calif.). Analyses of cell motility were performed on images taken every 5 min by tracing the motility paths of the center of individual nuclei defined as the crossing point of the longest and shortest axes of each nucleus of the cells. Motility rate was defined by the algebraic sum of the 2-dimensional migration distances every 25 min using NIH image. Migration of at least 12 cells was analyzed for each experimental condition. Excluded from this analysis were dividing cells thought to be in mitotic phase during the recording period and rounded cells considered to be losing viability. The statistical significance of findings was assessed according to the unpaired t test.

Antisense oligonucleotides
The oligonucleotides were purchased from GIBCO BRL. The antisense oligonucleotide sequences were anti-p21 (5'-TCCCCAGCCGGTTCTGACAT-3') and anti-p27 (5'-GACACTCTCACGTTTGACAT-3'), which are complementary to the region around the initiation codon of p21 (22) and p27 (23), respectively. We used p21 sense (5'-AGGGGTCGGCCAAGACTGTA-3') and p27 sense (5'-CTGTGAGAGTGCAAACTGTA-3') oligonucleotides as controls. To determine the effect of antisense oligonucleotides on tumor cells, oligonucleotides (100 µM) and lipofectamine were incubated at 37°C for 10 min. The oligonucleotide-lipofectamine mixture was diluted with serum-containing medium and added to the cells each day, giving a final concentration of 1.0 µM. Immunoblotting assay was performed to investigate whether antisense effectively inhibits the expression of target protein. After 4 days, cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) (Boehringer-Mannheim) colorimetric assay as previously described (28). Tumor cells were also stained with Hoechst 33258 to detect apoptotic morphology as described above. The statistical significance of findings was assessed using the unpaired t test.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Effect of antisense human telomerase on malignant glioma cells
As shown in Fig. 1, telomerase activity was clearly detected in parental U251-MG cells. Four clones (A to D) transfected with the pBabe anti-telo plasmid had undetectable levels of telomerase activity at 15 ( Fig. 1) and 60 doublings (data not shown) after transfection, whereas telomerase activity was retained in U251-MG cells transfected with the control pBabe-puro plasmid. Internal standard bands were detected in all samples, which excludes the possibility of false-negative results due to Taq polymerase inhibitor. Until 30 doublings, all clones retained the same morphology and growing pattern as parental U251-MG cells (data not shown). After 30 doublings, apoptotic cell death was morphologically demonstrated in about 40% of cells from each clone over a period of 1 wk ( Fig. 2A). DNA prepared from clones A and B at 30 doublings after transfection showed the typical nucleosome spacing ladder on agarose gel indicative of apoptosis ( Fig. 2B). Parental U251-MG cells and clones A and B at 15 and 60 doublings after transfection did not display DNA fragmentation ( Fig. 2B). Clones C and D showed a similar tendency. To clarify the molecular mechanisms of apoptosis in clones (A to D) at 30 doublings after transfection, we evaluated the expression of the protease, ICE. All clones (A to D) without telomerase activity exhibited an enhanced level of ICE protein (10 to 12-fold) at 30 doublings after transfection ( Fig. 3), but did not show enhanced ICE protein prior to 30 doublings after transfection (data not shown). ICE protein was undetected in parental U251-MG and U251-MG cells transfected with the control vector ( Fig. 3). The enhanced expression of ICE in all clones (A to D) was retained at 60 doublings after transfection (data not shown).

View larger version (34K): [in this window] [in a new window]   Figure 1. Telomerase activity in parental U251-MG cells, U251-MG transfected with the control vector, and four clones (A to D) transfected with the pBabe anti-telo plasmid. Cell extracts were incubated with the TS oligonucleotide in the presence of dNTPs and [-32P]dCTP; after addition of the CX primer, elongated TS oligonucleotides were amplified by PCR in the presence of an internal TRAP assay standard (ITAS). Reaction products were resolved on a 10% polyacrylamide gel. Extracts of four clones (A to D) were isolated at 15 doublings after transfection, respectively. Results shown are representative of two independent experiments.

View larger version (218K): [in this window] [in a new window]   Figure 2. Apoptotic features. A) Hoechst 33258 staining. Clone A cells at 30 doublings after transfection with the pBabe anti-telo plasmid were fixed with 1% formaldehyde/0.2% glutaraldehyde and stained with Hoechst 33258 (x200). B) DNA fragmentation in agarose gel. Fragmented DNA was isolated and electrophoresed in a 1.5% agarose gel containing 0.5 µg/ml ethidium bromide. Molecular weight standards of multiples of 123 bp DNA Ladder (GIBCO BRL, Tokyo) are shown in lane 1. Lane 2, U251-MG cells; lanes 3–5, clone A at 15, 30, and 60 doublings after transfection with the pBabe anti-telo plasmid; lanes 6–8, clone B at 15, 30, and 60 doublings after transfection.

View larger version (30K): [in this window] [in a new window]   Figure 3. Expression of ICE protein in clones transfected with the pBabe anti-telo plasmid. Immunoblotting using anti-ICE antibody was performed with equal amounts of proteins isolated from parental U251-MG cells, U251-MG transfected with the control vector, and four clones (A–D) at 30 doublings after transfection. The anti-actin antibody was used for protein loading equivalence. Results are representative of two independent experiments.

These results suggested that inhibition of telomerase enhanced the expression of ICE and subsequently induced apoptosis in some subpopulations of clones (A to D) without telomerase activity. The mechanisms by which other subpopulations of four clones (A to D) escaped apoptosis while expressing high level of ICE protein, however, were unclear. The nonapoptotic fractions of each clone proliferated in culture, but DNA synthesis was significantly inhibited compared to parental U251-MG cells ( Table 1). Inhibition of telomerase resulted in a decrease in the percentage of cells in S phase and an increase in the percentage of cells in G₂/M phase in all clones (A to D) compared to parental U251-MG cells. These results indicate that antisense human telomerase reduced cell cycling and caused an accumulation of cells in G₂/M in clones (A to D) that had escaped apoptotic cell death. To examine the effect of antisense telomerase on the invasive activity of tumor cells, a Matrigel assay was performed. After 72 h of culture, parental U251-MG cells began to divide vigorously and extended the cell processes throughout the gel ( Fig. 4). After a 7 day culture, parental U251-MG cells invaded the Matrigel and formed spherical and multiple aggregates. In contrast, cell numbers of clone A did not increase ( Fig. 4). Other clones (B to D) also showed a similar tendency (data not shown). Since four clones showed a similar tendency, clones A and B were used for additional studies. Clones A and B were also tested for tumorigenicity in athymic nude mice by subcutaneous injection. After inoculation of 2.5 x 10⁶ cells, parental U251-MG cells formed palpable masses in all nude mice (4/4) by 7 days. The mean mass volume was 101 ± 5.5 m³ 3 wk after inoculation. In contrast, clones A and B failed to form tumors (0/4). These results indicate that invasive properties of U251-MG cells were almost completely inhibited after treatment with antisense telomerase.

View larger version (152K): [in this window] [in a new window]   Figure 4. Morphological changes in parental U251-MG cells and clone A at 40 doublings after transfection with the pBabe anti-telo plasmid in Matrigel 3-dimensional cultures. (x250). a) U251-MG cells, 3 days; b) U251-MG cells, 7 days; c) clone A, 3 days; d) clone A, 7 days. Results are representative of two independent experiments.

Differentiation of malignant glioma cells
Inhibition of telomerase directed U251-MG cells to two distinct pathways: apoptosis and a nonapoptotic state of reduced growth and invasive ability. To characterize the phenotype of nonapoptotic cells exhibiting undetectable telomerase, expression of GFAP was analyzed. GFAP is an astrocyte-specific intermediate filament and is related to differentiation in malignant gliomas (33, 34). The antisense telomerase-transfected cell clones (A and B) demonstrated elevated GFAP expression when compared to parental U251-MG cells or U251-MG cells transfected with the control vector ( Fig. 5). Densitometric scans of the immunoblot showed that the expression of GFAP was remarkably increased (five- and sixfold) in clones A and B, respectively, at 40 doublings after transfection compared to parental U251-MG cells.

View larger version (25K): [in this window] [in a new window]   Figure 5. Expression of GFAP in parental U251-MG cells, U251-MG transfected with the control vector, and clones A and B at 40 doublings after transfection with the pBabe anti-telo plasmid. Immunoblotting using anti-GFAP antibody was performed with equal amounts of protein. The anti-actin antibody was used for protein loading equivalence. Results are representative of two independent experiments.

Because cell motility may decrease due to differentiation of tumor cells (35), the motility rates of U251-MG and clone A were also compared. The movement of 12 randomly chosen U251-MG cells and clone A at 40 doublings after transfection was followed by tracking the movement at 5 min intervals. Dramatic differences in the movement of the two cell populations are illustrated by the tracks of representative cells in Fig. 6A. U251-MG cells had high motility and some cells migrated relatively long distances, whereas clone A cells had less motility. As shown in Fig. 6B, the motility of clone A was significantly decreased compared to parental U251-MG cells (P<0.0001). These results suggest that inhibition of telomerase induced differentiation in subpopulations that had escaped from apoptosis.

View larger version (42K): [in this window] [in a new window]   Figure 6. Effect of inhibition of telomerase on motility of malignant glioma cells. For time lapse videomicrography, a digital image was taken every 5 min for 24 h with a charge-coupled device camera. A) The paths taken by 12 randomly chosen parental U251-MG cells and clone A at 40 doublings after transfection for 24 h are represented. Shown are the starting () and finishing () positions of cells. Bar represents 250 µm. B) Motility rate. The total movement distance of 12 randomly chosen parental U251-MG cells and clone A at 40 doublings after transfection was calculated as the sum of distances traveled during each 25 min interval over the 425 min period. 182 pixels correspond to 0.5 mm. Values are means with standard errors of motility rates of 12 cells. The data were collected from two independent experiments. *Significance at P<0.0001 as compared with U251-MG cells.

Expression of cyclin-dependent kinase inhibitors p21 and p27
CDKIs are shown to be associated with differentiation in some cell types (36–38). To determine whether inhibition of telomerase activity affected the expression of CDKIs (p16, p21, and p27) in tumor cells, immunoblotting assays were performed. As shown in Fig. 7, the expression of p16 was very low or undetectable in parental U251-MG, U251-MG transfected with the control vector and in clones A and B. Protein levels of both p21 and p27 were remarkably enhanced (five-to sixfold) in clones A and B, respectively, at 40 doublings after transfection when compared to parental U251-MG cells ( Fig. 7). These nonapoptotic clones retained the enhanced expression of p21 and p27 at 60 doublings after transfection (data not shown). Taking these findings together with the results of ICE expression, we hypothesized that enhanced expression of p21 or p27 might protect differentiating cells from ICE-associated apoptosis. To test this hypothesis, we used p21 and p27 antisense oligonucleotides to block expression of p21 and p27, respectively. As judged by immunoblotting assays, expressions of p21 and p27 in clones A ( Fig. 8) and B (data not shown) were reduced to undetectable levels by antisense treatment for 4 days. As shown in Table 2, the antisense oligonucleotide p21 had no apparent effect on the cell viability or nuclear morphology of parental U251-MG cells and clones A and B. Treatment of clones A and B, but not parental U251-MG cells, with antisense p27 inhibited cell viability and induced apoptosis ( Table 2). These results suggested that enhanced expression of p27, but not p21, directly protected differentiating glioma cells from apoptosis.

View larger version (25K): [in this window] [in a new window]   Figure 7. Expression of cyclin-dependent kinase inhibitors (CDKIs). Immunoblotting using anti-p16, p21, or p27 antibody was performed with equal amounts of protein isolated from parental U251-MG, U251-MG transfected with the control vector, and clones A and B at 40 doublings after transfection with the pBabe anti-telo plasmid. The anti-actin antibody was used for protein loading equivalence. Results are representative of two independent experiments.

View larger version (17K): [in this window] [in a new window]   Figure 8. Effect of antisense p21 or p27 oligonucleotide on expression of p21 or p27 protein in clone A. Clone A cells at 40 doublings after transfection with the pBabe anti-telo plasmid were used. Tumor cells were seeded at 105 cells/ml in 6-well plates and incubated overnight. Each oligonucleotide (100 µM) and lipofectamine was incubated at 37°C for 10 min. The oligonucleotide-lipofectamine mixture was diluted with serum-containing medium and added to the cells each day (1.0 µM). After 4 days of treatment, immunoblotting using anti-p21 or p27 antibody was performed with equal amounts of proteins isolated from untreated and treated clone A cells. The anti-actin antibody was used for protein loading equivalence. Results are representative of two independent experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Little is known about the specific links between telomerase activity, apoptosis, and differentiation. The studies described here provide several intriguing clues to this relationship. First, some subpopulations of glioma cells transfected with the pBabe anti-telo plasmid entered an apoptotic crisis over a period of 1 wk after 30 doublings. A key signal for apoptosis may have been provided by the markedly enhanced expression of ICE. Second, the expression of CDKIs (p21 and p27) was enhanced in tumor cells that escaped from apoptosis. These nonapoptotic cells appeared to undergo differentiation, but p27 antisense treatment caused them to undergo apoptotic cell death. From these findings, we hypothesize that inhibition of telomerase in tumor cells exhibiting telomerase activity triggers two distinct pathways, apoptosis and differentiation, which are regulated through enhanced expression of ICE or CDKIs. Inhibition of telomerase has recently been shown to shorten telomere length and, subsequently, induce cell death in HeLa cells after 23 to 26 doublings (17); however, no data were presented concerning the percentage of tumor cells undergoing cell death or whether differentiation was observed. The differences between such findings and our results may depend on the level of telomerase inhibition or the expression ratio of ICE to CDKIs in tumor cells. Since the same antisense vector against human telomerase RNA (17) was used in this study, it is expected that the telomere length of U251-MG cells tested by us was shortened by inhibition of telomerase function.

Telomerase activity has been detected frequently in various tumor types (11, 39, 40). Avoidance of telomeric shortening by expression of telomerase may contribute to the immortal phenotype. A favorable prognosis appears to be associated with low or no telomerase activity in some tumors (14, 15). On the other hand, some immortal cell lines without detectable telomerase activity have been described that were characterized by long and heterogenous telomeres (41, 42). These observations might indicate the presence of a telomerase-independent mechanism for telomere length maintenance in these tumors.

After 30 doublings, approximately 40% of U251-MG cells transfected with the pBabe anti-telo plasmid were induced to undergo an apoptotic crisis. Apoptosis is an essential physiological process that is regulated genetically. A variety of stimuli are known to induce or suppress apoptosis (43–47). Tumor cells may undergo apoptosis via p53-dependent or p53-independent pathways (20, 48–52). Since U251-MG cells express mutant p53 (25), antisense telomerase may induce p53-independent apoptosis in these cells. Recently, we have demonstrated that overexpression of ICE gene induces apoptosis in malignant glioma cells regardless of p53 status (20). ICE or ICE-related proteases (ICE family) have been identified as regulators of apoptosis in several cell types (19–21, 53). The specific substrate that the ICE family acts upon to induce apoptosis is presently unknown, however. ICE may cause cell death directly by proteolytically cleaving proteins that are essential for cell viability, and the activation of ICE-like ptoteases may initiate a final common pathway of apoptosis (19). The present studies indicate that inhibition of telomerase enhanced the expression of ICE in tumor cells that were surviving or undergoing apoptosis. We therefore raise the possibility that telomerase may inhibit ICE protein expression in tumor cells and subsequently confer a resistance to apoptosis.

The direct inhibition of telomerase with antisense effectively suppressed proliferative growth, reduced tumorigenicity in nude mice, enhanced the expression of GFAP, and decreased the motility of glioma cells that escaped from apoptosis. GFAP is an astrocyte-specific intermediate filament thought to provide structural support to normal astrocytes. The expression of GFAP in astrocytomas induced growth inhibition and stellate process formation indicative of a more differentiated astrocytic phenotype (33, 34). Also, the motility of glioma may be related to malignancy because suppression of tumorigenic potential decreases the motility of glioma cells (35). Our results therefore suggest that inhibition of telomerase activity induces differentiation in subpopulations of malignant glioma cells that escape from apoptotic crisis, raising the possibility of a link between telomerase activity and differentiation. In human leukemia cells, telomerase activity was inhibited when terminal differentiation was induced by diverse agents (54, 55). Such findings may be compatible with our results, although clones (A to D) presented here did not reach the terminal differentiation because they could divide even at 60 doublings after transfection. It may be difficult to understand why our clones without telomerase activity could grow slowly, but continuously, at 60 doublings. The enhanced expression of p21 and p27 might affect their growth pattern.

Progression through the cell cycle is mediated by sequential formation, activation, and subsequent inactivation of a series of CDK complexes (56–58). In mammals, there are two known groups of CDKI proteins: the KIP/CIP family (p21, p27, and p57), which can bind to and inhibit a broad range of CDK-cyclin complexes (22–24, 59); and the INK family (p15, p16, and p18), which are specific inhibitors of the CDK4/CDK6 kinases (60, 61). Recent demonstrations suggest that the up-regulation of p21 or p27 is the major determinant for differentiation in neuroblastoma (36) or myocytes (38). The present data showing that expression of p21 and p27 may be related to differentiation are consistent with these observations and therefore support the possibility of a link between telomerase activity and CDKI expression. Telomerase activity is likely to be regulated in a cell cycle-dependent manner (62). As the cell progresses through the cell cycle, telomerase activity becomes maximal in S phase, with barely detectable levels observed at G₂/M phase. These observations may be consistent with the present results showing that inhibition of telomerase leads to decreased S phase and increased G₂/M phase cells. It is surprising that treatment of clones A and B with p27 antisense oligonucleotides, but not p21, induced apoptosis. Here, p27 is required for survival of differentiating glioma cells. It may be difficult to simply conclude that the enhanced p27 expression or changes in cell cycle may have arisen directly as a consequence of diminished telomerase function. It is more likely we have simply selected for cells capable of surviving the growth crisis, which may therefore be slow-growing. It thus seems necessary to examine possible altered cell cycle parameters in cells before the crisis or to use an inducible antisense vector. Additional studies are needed to determine the roles of p21 and p27 in antisense telomerase-associated differentiation.

It has recently been demonstrated that mice in which the telomerase RNA component was destroyed by knockout were viable for up to six generations and that their cells were capable of ras-mediated transformation and immortalization; eventually, however, as telomeres became shorter, the cells or animals were increasingly subject to dramatic chromosomal rearrangements that would eventually limit viability in the organism (63). These results seem contrary to our findings. However, the fact that telomerase is activated in mouse tumors in vivo (64–67) raises the question of why telomerase is activated in mouse tumors if it is not essential for tumor growth. Although mice provide a powerful tool for understanding cancer progression, some significant differences between human and mouse biology may have implications for tumor growth (63). It is possible that telomerase may be required for the growth of telomerase-positive human tumors. More recently, we have demonstrated that inhibition of telomerase might represent a promising strategy for treating malignant gliomas due not only to induction of apoptosis (68) but also chemosentitization (26), although the precise mechanisms remain unclear. In summary, we hypothesize that telomerase may play an important role in the malignancy not only by conferring invasive activity, but also by affording protection from apoptosis as well as by preventing differentiation. After a shutdown of telomerase, ICE or CDKIs may constitute part of the equation that determines whether a tumor cell dies or continues to grow slowly in a more differentiated state. Telomerase represents a viable target for the treatment of malignant tumors with telomerase activity; understanding the mechanisms of telomerase inhibition may enhance this novel therapeutic approach.

	ACKNOWLEDGMENTS

 
We thank Dr. Dennis W. Stacey and Dr. Tony Hunter for helpful suggestions and Geron Corporation for antisense telomerase vector (p-10–3-hTR). This study was supported in part by the John Gagliarducci Fund (S.K.), in part by National Institutes of Health Grant (R01NS-33932) (B.P.B.), and by funds from Atlantic Pharmaceuticals, Inc. (J.K.C.).

	FOOTNOTES

 
¹ Correspondence: Department of Neurosurgery/S80, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA. E-mail: kondos{at}cesmtp.ccf.org

² Abbreviations: ICE, interleukin-1-converting enzyme; CDKI, cyclin-dependent kinase inhibitor; DMEM, Dulbecco's modified Eagle's medium; PCR, polymerase chain reaction; GFAP, glial fibrillary acidic protein.

Received for publication October 30, 1997. Accepted for publication February 10, 1998.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Schoenberg, B. S. (1983) Oncology of the Nervous System (Walker, M. D., ed) pp. 1–30, Nifhoff, Boston
2. Collins, M. K. L., Sinnett-Smith, J. W., and Rozengurt, E. (1983) Platelet-derived growth factor treatment decreases the affinity of the epidermal growth factor receptors of Swiss 3T3 cells. J. Biol. Chem. 258, 11689–11693[Abstract/Free Full Text]
3. Libermann, T. A., Nusbaum, H. R., Razon, N., Kris, R., Lax, I., Soreq, H., Whittle, N., Waterfield, M. D., Ullrich, A., and Schlessinger, J. (1985) Amplification, enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumors of glial origin. Nature (London) 313, 144–147[Medline]
4. Sidransky, D., Mikkelsen, T., Schwechheimer, K., Rosenblum, M. L., Cavene, W. K., and Vogelstein, B. (1992) Clonal expansion of p53 mutant cells is associated with brain tumor progression. Nature (London) 355, 846–847[Medline]
5. Mehle, C., Piatyszek, M. A., Ljungberg, B., Shay, J. W., and Roos, G. (1996) Telomerase activity in human renal cell carcinoma. Oncogene 13, 161–166[Medline]
6. Blackburn, E. H. (1991) Structure and function of telomeres. Nature (London) 350, 569–573[Medline]
7. de Lange, T. (1994) Activation of telomerase in a human tumor. Proc. Natl. Acad. Sci. USA 91, 2882–2885[Free Full Text]
8. Harley, C. B., Futcher, A. B., and Greider, C. W. (1990) Telomeres shorter during aging of human fibroblasts. Nature (London) 345, 458–460[Medline]
9. Hastie, N. D., Dempster, M., Dunlop, M. G., Thompson, A. M., Geen, D. K., and Allshire, R. C. (1990) Telomere reduction in human colorectal carcinoma and with aging. Nature (London) 346, 866–868[Medline]
10. Greider, C. W., and Blackburn, E. H. (1985) Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 43, 405–413[Medline]
11. Counter, C. M., Avilion, A. A., LeFeuvre, C. E., Stewart, N. G., Greider, C. W., Harley, C. B., and Bacchetti, S. (1992) Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO J. 11, 1921–1929[Medline]
12. Kim, N. W., Piatyszek, M. A., Prowse, K. R., Harley, C. B., West, M. D., Ho, P. L. C., Coviello, G. M., Wright, W. E., Weinrich, S. L., and Shay, J. W. (1994) Specific association of human telomerase activity with immortal cell lines and cancer. Science 266, 2011–2015[Abstract/Free Full Text]
13. Broccoli, D., Young, J. W., and de Lange, T. (1995) Telomerase activity in normal and malignant hematopoietic cells. Proc. Natl. Acad. Sci. USA 92, 9082–9086[Abstract/Free Full Text]
14. Hiyama, K., Hiyama, E., Ishioka, S., Yamakido, M., Inai, K., Gazdar, A. F., Piatyszek, M. A., and Shay, J.W. (1995) Telomerase activity in small-cell and non-small-cell lung cancers. J. Natl. Cancer Inst. 87, 895–902[Abstract/Free Full Text]
15. Hiyama, E., Yokoyama, T., Tatsumoto, N., Hiyama, K., Imamura, Y., Murakami, Y., Kodama, T., Piatyszek, A. P., Shay, J. W., and Matsuura, Y. (1995) Telomerase activity in gastric cancer. Cancer Res. 55, 3258–3262[Abstract/Free Full Text]
16. Chiu, C. P., Dragowska, W., Kim, N. W., Vaziri, H., Yui, J., Thomas, T. E., Harley, C. B., Lansdorp, P. M. (1996) Differential expression of telomerase activity in hemopoietic progenitors from adult human bone marrow. Stem Cells 14, 239–248[Abstract]
17. Feng, J., Funk, W. D., Wang, S.-S., Weinrich, S. L., Avilion, A. A., Chiu, C.-P., Adams, R. R., Chang, E., Allsopp, R. C., Yu, J., Le, S., West, M. D., Harley, C. B., Andrews, W. H., Greider, C.W., and Villeponteau, B. (1995) The RNA component of human telomerase. Science 269, 1236–1241[Abstract/Free Full Text]
18. Yuan, J., Shaham, S., Ledoux, S., Ellis, H. M., and Horvitz, H. R. (1993) The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1ß-converting enzyme. Cell 75, 641–652[Medline]
19. Miura, M., Zhu, H., Rotello, R., Hartwieg, E. A., and Yuan, J. (1993) Induction of apoptosis in fibroblasts by IL-1ß-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3. Cell 75, 653–660[Medline]
20. Kondo, S., Barna, B. P., Morimura, T., Takeuchi, J., Yuan, J., Akbasak, A., and Barnett, G. (1995) H. Interleukin-1ß-converting enzyme (ICE) mediates cisplatin-induced apoptosis in malignant glioma cells. Cancer Res. 55, 6166–6171[Abstract/Free Full Text]
21. Kondo, S., Kondo, Y., Yin, D., Barnett, G. H., Kaakaji, R., Peterson, J. W., Morimura, T., Kubo, H., Takeuchi, J., and Barna, B. P. (1996) Involvement of interleukin-1ß-converting enzyme in apoptosis of bFGF-deprived murine aortic endothelial cells. FASEB J. 10, 1192–1197[Abstract]
22. El-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent, J. M., Lin, D., Mercer, W. E., Kinzler, K. W., and Vogelstein, B. (1993) WAF1, a potential mediator of p53 tumor suppression. Cell 75, 817–825[Medline]
23. Polyak, K., Lee, M.-H., Erdjument-Bromage, H., Koff, A., Roberts, J. M., Tempst, P., and Massagu, J. (1994) Cloning of p27^Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell 78, 59–66[Medline]
24. Toyoshima, H., and Hunter, T. (1994) p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21. Cell 78, 67–74[Medline]
25. Van Meir, E. G., Kikuchi, T., Tada, M., Li, H., Diserens, A. C., Wojcik, B. E., Huang, H. J. S., Friedmann, T., de Tribolet, N., and Cavenee, W. K. (1994) Analysis of the p53 gene and its expression in human glioblastoma cells. Cancer Res. 54, 649–652[Abstract/Free Full Text]
26. Kondo, Y., Kondo, S., Tanaka, Y., Haqqi, T., Barna, B. P., and Cowell, J. K. (1998) Inhibition of telomerase increases the susceptibility of human malignant glioblastoma cells to cisplatin-induced apoptosis. Oncogene In press
27. Kondo, S., Barnett, G. H., Hara, H., Morimura, T., and Takeuchi, J. (1995) MDM2 protein confers the resistance of human glioblastoma cell line to cisplatin-induced apoptosis. Oncogene 10, 2001–2006[Medline]
28. Yin, D., Kondo, S., Takeuchi, J., and Morimura, T. (1994) Induction of apoptosis in murine ACTH-secreting pituitary adenoma cells by bromocriptine. FEBS Lett. 339, 73–75[Medline]
29. Kondo, S., Morimura, T., Barnett, G. H., Kondo, Y., Peterson, J. W., Kaakaji, R., Takeuchi, J., Toms, S.A., Liu, J., Werbel, B., and Barna, B. P. (1996) The transforming activities of MDM2 in cultured neonatal rat astrocytes. Oncogene 13, 1773–1779[Medline]
30. Barna, B. P., Estes, M. L., Pettay, J., Iwasaki, K., Zhou, P., and Barnett, G. H. (1995) Human astrocyte growth regulation: interleukin-4 sensitivity and receptor expression. J. Neuroimmunol. 60, 75–81[Medline]
31. Kondo, S., Yin, D., Morimura, T., Kubo, H., Nakatsu, S., and Takeuchi, J. (1995) Combination therapy with cisplatin and nifedipine induces apoptosis in cisplatin-sensitive and cisplatin-resistant human glioblastoma cells. Br. J. Cancer. 71, 282–289[Medline]
32. Fox, P. L., Sa, G., Dobrowolski, S. F., and Stacey, D. W. (1993) The regulation of endothelial cell motility by p21 ras. Oncogene 9, 3519–3526
33. Rutka, J. T., and Smith, S. L. (1993) Transfection of human astrocytoma cells with glial fibrillary acidic protein complementary DNA: analysis of expression, proliferation, and tumorigenicity. Cancer Res. 53, 3624–3631[Abstract/Free Full Text]
34. Rutka, J. T., Hubbard, S. L., Fukuyama, K., Matsuzawa, K., Dirks, P. B., and Becker, L. E. (1994) Effects of antisense glial fibrillary acidic protein complementary DNA on the growth, invasion, and adhesion of human astrocytoma cells. Cancer Res. 54, 3267–3272[Abstract/Free Full Text]
35. Okada, H., Yoshida, J., Sokabe, M., Wakabayashi, T., and Hagiwara, M. (1996) Suppression of CD44 expression decreases migration and invasion of human glioma cells. Int. J. Cancer. 66, 255–260[Medline]
36. Kranenburg, O., Scharnhorst, V., Van der Eb, A. J., Zantema, A. (1995) Inhibition of cyclin-dependent kinase activity triggers neuronal differentiation of mouse neuroblastoma cells. J. Cell Biol. 131, 227–234[Abstract/Free Full Text]
37. Liu, M., Lee, M.-H., Cohen, M., Bommakanti, M., and Freedman, L. P. (1996) Transcriptional activation of the Cdk inhibitor p21 by vitamin D₃ leads to the induced differentiation of the myelomonocytic cell line U937. Genes & Dev. 10, 142–153[Abstract/Free Full Text]
38. Wang, J., and Walsh, K. (1996) Resistance to apoptosis conferred by Cdk inhibitors during myocyte differentiation. Science 273, 359–361[Abstract]
39. Counter, C. M., Gupta, J., Harley, C. B., Leber, B., and Bacchetti, S. (1995) Telomerase activity in normal leukocytes and in hematologic malignancies. Blood 85, 2315–2320[Abstract/Free Full Text]
40. Chadeneau, C., Hay, K., Hirte, H. W., Gallinger, S., and Bacchetti, S. (1995) Telomerase activity associated with acquisition of malignancy in human colorectal cancer. Cancer Res. 55, 2533–2536[Abstract/Free Full Text]
41. Rogan, E. M., Bryan, T. M., Hukku, B., Maclean, K., Chang, A. C., Moy, E. L., Englezou, A., Warneford, S. G., Dalla-Pozza, L., and Reddel, R. R. (1995) Alterations in p53 and p16^INK4 expression and telomere length during spontaneous immortalization of Li-Fraumeni syndrome fibroblasts. Mol. Cell. Biol. 15, 4745–4753[Abstract]
42. Strahl, C., and Blackburn, E. H. (1996) Effect of reserve transcriptase inhibitors on telomere length and telomerase activity in two immortalized human cell lines. Mol. Cell. Biol. 16, 53–65[Abstract]
43. Wyllie, A. H. (1987) Cell death. Int. Rev. Cytol. 17, 755–785
44. Kaufman, S. H. (1989) Induction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etooside, camptotecin, and other cytotoxic anti-cancer drugs; a cautionary note. Cancer Res. 49, 5870–5878[Abstract/Free Full Text]
45. Eastman, A. (1990) Activation of programmed cell death by anticancer agents: cisplatin as a model system. Cancer Cells 2, 275–280[Medline]
46. Kondo, S., Yin, D., Morimura, T., Oda, Y., Kikuchi, H., and Takeuchi, J. (1994) Transfection with a bcl-2 expression vector protects transplanted bone marrow from chemotherapy-induced myelosuppression. Cancer Res. 54, 2928–2933[Abstract/Free Full Text]
47. Kondo, S., Yin, D., Aoki, T., Takahashi, J. A., Morimura, T., and Takeuchi, J. (1994) bcl-2 gene prevents apoptosis of basic fibroblast growth factor-deprived murine aortic endotheliac cells. Exp. Cell Res. 213, 428–432[Medline]
48. Levine, A. J., Momand, J., and Finlay, C. A. (1991) The p53 tumour suppressor gene. Nature (London) 351, 453–456[Medline]
49. Yonish-Rouach, E., Resnitzky, D., Lotem, J., Sachs, L., Kimchi, A., and Oren, M. (1991) Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6. Nature (London) 352, 345–340[Medline]
50. Clarke, A. R., Purdie, C. A., Harrison, D. J., Morris, R. G., Bird, C. C., Hooper, M. L., and Wyllie, A. H. (1993) Thymocyte apoptosis induced by p53-dependent and independent pathways. Nature (London) 362, 849–852[Medline]
51. Fritsche, M., Haessler, C., and Brandner, G. (1993) Induction of nuclear accumulation of the tumor-suppressor protein p53 by DNA-damaging agents. Oncogene 8, 307–318[Medline]
52. Lowe, S. W., Earl-Ruley, H., Jacks, T., and Housman, D. E. (1993) p53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell 74, 957–967[Medline]
53. Wang, L., Miura, M., Bergeron, L., Zhu, H., and Yuan, J. (1994) Ich-1, an Ice/ced-3-related gene, encodes both positive and negative regulators of programmed cell death. Cell 78, 739–750[Medline]
54. Sharma, H. W., Sokoloski, J. A., Perez, J. R., Maltese, J. Y., Sartorelli, A. C., Stein, C. A., Nichols, G., Khaled, Z., Telang, N.T., and Narayanan, R. (1995) Differentiation of immortal cells inhibits telomerase activity. Proc. Natl. Acad. Sci. USA 92, 12343–12346[Abstract/Free Full Text]
55. Bestilny, L. J., Brown, C. B., Miura, Y., Robertson, L. D., and Riabowol, K. T. (1996) Selective inhibition of telomerase activity during terminal differentiation of immortal cell lines. Cancer Res. 56, 3796–3802[Abstract/Free Full Text]
56. Reed, S. I. (1992) The role of p34 kinases in the G₁ to S-phase transition. Annu. Rev. Cell. Biol. 8, 529–561
57. Pines, J. (1993) Cyclins and cyclin-dependent kinases; take your partners. Trends Biochem. Sci. 18, 195–197[Medline]
58. Sherr, C. J. (1993) Mammalian G₁ cyclins. Cell 73, 1059–1065[Medline]
59. Lee, M., Reynisdottir, I., and Massague, J. (1995) Cloning of p57^Kip2, a cyclin-dependent kinase inhibitor with unique domain structure and tissue distribution. Genes & Dev. 9, 639–649[Abstract/Free Full Text]
60. Serrano, M., Hannon, G., and Beach, D. (1993) A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature (London) 366, 704–707[Medline]
61. Xiong, Y., Zhang, H., Beach, D. (1993) Subunit rearrangement of the cyclin-dependent kinases is associated with cellular transformation. Genes & Dev. 7, 1572–1583[Abstract/Free Full Text]
62. Zhu, X., Kumar, R., Mandal, M., Sharma, N., Sharma, H. W., Dhingra, U., Sokoloski, J. A., Hsiano, R., and Narayanan, R. (1996) Cell cycle-dependent modulation of telomerase activity in tumor cells. Proc. Natl. Acad. Sci. USA 93, 6091–6095[Abstract/Free Full Text]
63. Blasco, M. A., Lee, H.-W., Hande, M .P., Samper, E., Lansdorp, P. M., DePinho, R. A., and Greider, C. W. (1997) Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell 91, 25–34[Medline]
64. Bednarek, A., Budunova, I., Slaga, T. J., and Aldaz, C. M. (1995) Increased telomerase activity in mouse skin pre-malignant progression. Cancer Res. 55, 4566–4569[Abstract/Free Full Text]
65. Chadeneau, C., Siegel, P., Harley, C. B., Muller, W., and Bacchetti, S. (1995) Telomerase activity in normal and malignant murine tissues. Oncogene 11, 893–898[Medline]
66. Blasco, M. A., Rizen, M., Greider, C. W., and Hanahan, D. (1996) Differential regulation of telomerase activity and telomerase RNA during multi-stage tumorigenesis. Nat. Genet. 12, 200–204[Medline]
67. Broccoli, D., Godley, L. A., Donehower, L. A., Varmus, H. E., and de Lange, T. (1996) Telomerase activation in mouse mammary tumors: lack of detectable telomere shortening and evidence for regulation of telomerase RNA with cell proliferation. Mol. Cell. Biol. 16, 3765–3772[Abstract]
68. Kondo, S., Kondo, Y., Li, G., Silverman, R.H., and Cowell, J. K. (1998) Targeted therapy of human malignant glioma in a mouse model by 2–5A antisense directed against telomerase RNA. Oncogene In press