The authors have no conflict of interest.
Azidothymidine Induces Apoptosis and Inhibits Cell Growth and Telomerase Activity of Human Parathyroid Cancer Cells in Culture†
Article first published online: 29 NOV 2004
Copyright © 2005 ASBMR
Journal of Bone and Mineral Research
Volume 20, Issue 3, pages 410–418, March 2005
How to Cite
Falchetti, A., Franchi, A., Bordi, C., Mavilia, C., Masi, L., Cioppi, F., Recenti, R., Picariello, L., Marini, F., Del Monte, F., Ghinoi, V., Martineti, V., Tanini, A. and Brandi, M. L. (2005), Azidothymidine Induces Apoptosis and Inhibits Cell Growth and Telomerase Activity of Human Parathyroid Cancer Cells in Culture. J Bone Miner Res, 20: 410–418. doi: 10.1359/JBMR.041123
- Issue published online: 4 DEC 2009
- Article first published online: 29 NOV 2004
- Manuscript Accepted: 15 OCT 2004
- Manuscript Revised: 10 SEP 2004
- Manuscript Received: 31 MAR 2004
- parathyroid cells;
- parathyroid cancer;
- parathyroid hormone;
- genetic research;
- genotype-phenotype correlation
Telomerase activity has been correlated to parathyroid carcinoma. Because its role in acquisition of a malignant phenotype by parathyroid cells is unclear, we treated telomerase-positive cultured human parathyroid cancer cells with the telomerase inhibitor AZT, evaluating cell telomerase activity, cytotoxic effects, growth, and morphological changes. In vitro exposure of these cells to AZT correlated with inhibition of cell proliferation.
Introduction: Parathyroid carcinoma represents an uncommon cause of primary hyperparathyroidism, whose spectrum of clinical presentation, degree of malignancy, and prognosis are difficult to be properly identified. Neck surgery, specifically an en bloc resection of primary tumor, is the only curative treatment. Alternatively, affected patients could undergo repetitive palliative surgical exeresis of metastatic nodules. It has been previously shown that telomerase activity is specifically present in parathyroid carcinoma cells, being absent in hyperplastic and adenomatous tissues. Thus, determination of telomerase activity could represent either a useful diagnostic molecular marker for human parathyroid carcinoma or a potential target for pharmacological intervention in a malignant neoplasia usually resistant to chemo- and radiotherapeutic interventions.
Materials and Methods: To further investigate the role of telomerase activity in acquisition of a malignant phenotype by parathyroid cells, we treated telomeric repeat amplification protocol-positive cultured human parathyroid cells with the telomerase inhibitor zidovudine, 3′-azido-3′deoxythymidine (AZT), evaluating cell telomerase activity, growth characteristics, potential cytotoxic effects, and morphological changes.
Results: Our findings indicate that in vitro exposure of human parathyroid cancer cells to AZT resulted in intracellular accumulation of AZT-monophosphate (AZT-MP) and inhibition of telomerase, which correlate with inhibition of human parathyroid cancer cell proliferation. Moreover, we also found that AZT induced an apoptotic rather than a necrotic type of cellular death. None of these effects were observed in human adenomatous parathyroid cells in culture.
Conclusions: Altogether these results indicate that AZT may be a highly effective agent against cancer parathyroid cells proliferation, which is an extremely important observation for a neoplasia which shows lack of response to classical pharmacological and physical antiblastic treatments.
ZIDOVUDINE, 3′-AZIDO-3′DEOXYTHYMIDINE (AZT),(1) is a chemotherapic molecule commonly used as antiviral agent, alone or in combination with other drugs, in the therapy of AIDS and human T-cell lymphotropic virus type I (HTLV)-I-associated adult T-cell leukemia/lymphoma.(2) Moreover, phase I and II clinical trials in gastrointestinal cancers showed that AZT contributed to regression of some tumors.(3–8)
AZT is a thymidine analog phosphorylated at the intracellular level to AZT-triphosphate (AZT-TP) by thymidine kinase enzyme, and in this form, it can be incorporated into viral DNA, acting as a false substitute for the viral RT, blocking chain elongation,(9) preferentially at the telomeric ends of chromosomes in cancer cells.(10) In combination with interferon-α, AZT is able to induce apoptosis in herpes virus-associated lymphomas,(11) and alone or in combination with other antimetabolites, it inhibits growth of human bladder, colon, and mammary cancer cells.(12,13)
Parathyroid carcinoma is an uncommon cause of primary hyperparathyroidism, and its spectrum of clinical presentation, degree of malignancy, and prognosis are difficult to identify.(14,15) An en bloc resection of the primary tumor is considered the only curative treatment. Alternatively, affected patients can undergo repetitive palliative surgical exeresis of metastatic nodules.
In a previous paper, we showed that telomerase activity, evaluated by telomeric repeat amplification protocol, is specifically present in parathyroid carcinoma cells compared with benign forms of parathyroid neoplasia.(16) Lack of telomerase activity in hyperplastic and adenomatous tissues was successively confirmed by others.(17–19) Thus, the determination of telomerase activity could represent either a useful diagnostic molecular marker for human parathyroid carcinoma or a potential target for pharmacological intervention in a malignant neoplasia usually resistant to chemo- and radiotherapeutic interventions.
To test this hypothesis, we decided to investigate the phenotype of telomeric repeat amplification protocol-positive cultured human parathyroid cancer (HPC) cells treated with the telomerase inhibitor, AZT, evaluating telomerase activity, growth characteristics, potential cytotoxic effects, and morphological changes.
MATERIALS AND METHODS
Primary cultures of human parathyroid cells
Human parathyroid malignant tissue was removed from two patients (a 72-year-old male [preparation I] and a 65-year-old female [preparation II]) who underwent surgery for metastatic parathyroid carcinoma. Histopathological features, such as nuclear atypia, numerous cells in mitosis, fibrous bands, and microvascular or capsular invasion were present. Parathyroid adenomatous tissue was obtained from three patients affected by benign primary hyperparathyroidism (a 66-year-old male [preparation III] and two female patients [preparations IV and V], who were 70 and 72 years old, respectively). Tumor samples were obtained in accordance with a protocol approved by the institutional review board for human studies; patients provided informed consent as dictated by this protocol. Cells were used within the first 10 days of primary culture, when >90% of the cell population showed immunostaining for parathyroid hormone (PTH).(20)
A fragment of the parathyroid pathological tissue was collected in sterile conditions and transported to the laboratory. Tissue was minced into small fragments and digested at 37°C for 2 h with 1.2 mg/ml collagenase type II (Sigma, Milan, Italy) in a mixture (1:1) of DMEM:Ham'sF-12 medium (DMEM:F-12 medium; Sigma). Digested tissue fragments were mechanically dispersed by aspiration into a pipette in DMEM:F-12 medium and successively centrifuged at 500g. The pellet was resuspended in DMEM:F-12 medium supplemented with 5% calf serum, 1% Nutridoma (Sigma), 1 mM CaCl2, 0.5 mM MgCl2, and antibiotics (growth medium). Cell suspension was filtered through 60 and 150 mesh screens (wire diameter, 0.191 and 0.066 mm, respectively; Sigma). The cells collected out of the 150 filters were distributed in plastic culture dishes in growth medium at a density of 5 × 105 cells/100-mm dish and cultured at 37°C in humidified 95% air/5% CO2 atmosphere.
Measurement of telomerase activity
Telomerase activity in intact cells was measured using a modified nonradioactive telomeric repeat amplification protocol and corrected for cell number.(21,22) Telomere length was measured by a solution hybridization-based method.(22) In brief, genomic DNA was isolated, and 10 μg of DNA was digested at 37°C overnight with 10 units each of HinfI/CpoI/HeaIII. The probe (TTAGGG)4 was labeled with γ-[32P]ATP with polynucleotide T4 kinase. Three nanograms of the probe was added to 2.5 μg of DNA solution. After denaturation at 98°C for 5 minutes, hybridization was performed at 55°C overnight. The resulting samples were electrophoresed on a 0.7% agarose gel. After drying under a vacuum without heating, the gel was exposed to a phosphorimage screen, and the results were analyzed using the area under the curve method by the ImageQuant software. The point representing 50% of the area under the curve was the mean telomere length. Genomic DNA was extracted from homogenated human parathyroid cultured cells after different times of exposure to AZT. Human malignant mammary cancer (MCF-7) cells (catalog no. HTB-22; ATCC) were used as a positive control.(23,24) HPC cells were treated with 100 μM AZT for different times, and total cells were collected and analyzed for the mean telomere length. All the experiments were carried out in triplicate.
Human parathyroid cells were also evaluated for mutations of the TP53 gene in exons 5–8. SSCP-screening kit by ANALITICA (Padova, Italy) was used to detect the presence of any kind of mutation in exons 5, 6, 7, and 8 of the TP53 gene, according to the manufacturer's instructions.
To examine both density and morphological features, HPC cells cultured in growth medium with and without 100 μM AZT for 72 h were observed under phase contrast light microscopy.
For light microscopy analysis, HPC cells were cultured on glass slides in the presence or in the absence of 100 μM AZT. Briefly, HPC cells cultured on glass slides were fixed in formaldehyde/glyceraldehyde (1:1) buffer for 3 minutes and washed twice in PBS.
For transmission electron microscopy (TEM) analysis, human parathyroid cells were cultured in growth medium with and without 100 μM AZT for 72 h, and the cells were collected by gentle scraping and centrifuged at 500g. The cell pellet was fixed in 4% cold glutaraldehyde in 0.1 M cacodylate buffer and postfixed in 1% osmium tetroxide in 0.1 M PBS at room temperature. The pellet was dehydrated in a graded acetone series, passed through propylene oxide, and embedded in epon 812. Ultrathin sections were cut, using a diamond knife, with a Leica Ultracut R microtome, mounted on formvar-coated Cu/Rh grids, stained with uranyl acetate and lead citrate, and observed with a Philips 410 LS transmission electron microscope.
Cell growth and viability
HPC cell proliferation was evaluated by [3H]thymidine uptake as previously described.(16) Briefly, HPC cells were incubated for 48 h under the indicated experimental conditions. In the last 6 h, [3H]thymidine was added, and cell mitogenesis was evaluated by the uptake of the labeled nucleotide.
Cell growth was measured by cell counting by plating HPC cells at a density of 3 × 105 cells/35-mm well under the indicated experimental conditions. After 96 h, HPC cells were detached with a trypsin solution (0.25% in PBS), and cell number was evaluated by a hemocytometer.
Cell viability was assessed by trypan blue dye uptake/exclusion assay. One drop of cell suspension was added to one drop of trypan blue dye solution (0.3% in PBS), and stained versus unstained cells were counted and expressed as percentage. Results were expressed as mean ± SD of triplicate experiments.
Antibody staining was performed on 1,000,000 cells for the conjugated primary antibody. Apoptosis was assayed with propidium iodide/annexin V staining. For propidium iodide/annexin V-FITC staining of apoptosis, cells were washed with annexin V FACS buffer (HBSS buffer with calcium, magnesium, sodium azide, and 0.5% BSA), incubated on ice with a 1:30 dilution of annexin V-FITC (Caltaq Laboratories, Burlingame, CA, USA) for 30 minutes, and washed in FACS buffer. Before FACS analysis, propidium iodide at 5 μg/ml (Sigma) was added to the cells and gently mixed. Stained cells were immediately analyzed by FACS.
HPC cells were plated at a density of 1 × 106 cells/35-mm dishes in growth medium for 24 h. Cells were exposed to DMEM/F-12 medium containing 0.01, 0.1, and 1.0 mM AZT for 6 h of incubation, media were collected and centrifuged at 2000g, and supernatants were frozen at −80°C. Intact human PTH (iPTH) released into the medium was evaluated by a commercially available IRMA kit for intact human PTH (Technogenetics, Milan, Italy). All experiments were carried out in triplicate and expressed as mean ± SD.
In vitro drug activity evaluation
Drug treatment was initiated after cells were allowed to attach to the growth surface. AZT (Sigma) is converted intracellularly to AZT-TP, which inhibits telomerase. Hence, we examined the effect of AZT only in intact cells. AZT effects measured at the indicated times at concentrations ranging from 0.1 μM to 1 mM were modifications of (1) morphological appearance of HPC cells both under optic and TE microscopy; (2) cell viability; (3) cell proliferative properties; (4) cell apoptosis; (5) iPTH release; and (6) telomerase activity. All experiments were carried out in triplicate and expressed as mean ± SD.
AZT phosphorylation assay
Intracellular levels of phosphorylated AZT were measured using published methods.(25) Cells were plated in 24-well plates at 5 × 105 cells/well and incubated with 5 μg/ml [3H]AZT (Sigma). After 24 h of incubation, cells were harvested and washed with PBS twice. Cell pellets were extracted with cold 65% methanol on ice for 30 minutes and spun at 1500 rpm for 5 minutes. The supernatants were collected and applied to HPLC for measurements of AZT-monophosphate (AZT-MP), because cytotoxicity of AZT correlates with AZT-MP levels, whereas anti-human immunodeficiency virus activity correlates with AZT-TP levels.(26,27)
Data were expressed as mean ± SD of triplicate experimental points. Statistical differences were analyzed using one-way ANOVA, and significance was evaluated by standard χ2 test using Statistica 5.1 (Statsoft, Tulsa, OK, USA).
The two HPC cell preparations showed comparable baseline telomere length (preparation I: 1675 ± 210 bp; preparation II: 2016 ± 307 bp). MCF-7 cells showed a telomere length of 2680 ± 218 bp. The three parathyroid adenomas resulted negative for telomerase activity (preparations III-V). None of the five samples (I-V) showed mutations in exons 5–8 of the TP53 gene.
Figure 1 shows the time-dependent telomerase inhibition by 0.1 mM AZT in HPC cells with significant effect after a 48-h incubation (preparations I and II). Results were corrected per cell number and expressed as mean ± SD of three different experiments.
Changes in functional and morphological features of HPC cells after AZT treatment
According to the trypan blue dye uptake/exclusion analysis, no cytotoxic effect was observed in cell preparations I and II at any of the AZT tested concentrations (data not shown).
Similarly, no significative changes in iPTH release were observed in untreated (preparation I: 100 ± 21 pg/106 cells; preparation II: 80 ± 18 pg/106 cells) and 0.1 mM AZT-treated HPC cells after a 6-h incubation (preparation I: 110 ± 17 pg/106 cells; preparation II: 85 ± 8 pg/106 cells). Lower (0.01 mM) and higher (1 mM) doses of AZT were similarly inert on iPTH release by HTC cells (data not shown).
Figures 2A and 2B compare the phase contrast morphology of HPC cells after 72-h exposure to 100 μM AZT. Treatment with AZT produced a change of the polygonal cell morphology of HPC cells into a spindle-shaped cellular profile. Moreover, the same treatment resulted in a reduction of cell number, with cells growing as sparse elements, as confirmed by cell counting of selected fields (preparation I: 65 ± 8% reduction versus control, p < 0.05; preparation II: 58 ± 4% reduction versus control, p < 0.01). Conversely, 0.01-1 mM AZT did not modify the phenotypic appearance of the telomerase-negative parathyroid adenomatous cells in preparations III-V (Figs. 2C and 2D).
Ultrastructurally, HPC cells were characterized by round to oval shape and irregular profile because of the presence of numerous interdigitating projections of the cell surface. The nucleus was round, centrally located, and contained moderately clumped and marginated chromatin and inconspicuous nucleoli. According to the cell cytoplasm content, it was possible to distinguish a spectrum of cell types, as seen in most parathyroid neoplasms. In untreated HPC cells cultures, cells presented features of the so-called “chief cells,” “inactive chief cells,” and “oxyphil cells.” Chief cells contained a well-developed Golgi apparatus, stacks of rough endoplasmic reticulum (RER) cisternae, elongated mitochondria, abundant glycogen particles, and several round to oblong electron dense endosecretory granules measuring 100–250 nm (Fig. 3A). Inactive chief cells were readily recognized by the presence of abundant cytoplasmic glycogen particles and more inconspicuous organelles and endosecretory granules (Fig. 3B). HPC cells treated with 100 μM AZT showed prevalent features of oxyphil cells, which were characterized by the presence of numerous mitochondria, a moderately developed secretory apparatus, and rare endosecretory granules (Fig. 3C). Transitional chief/oxyphil cells were also present, sharing ultrastructural features of both cell types (i.e., numerous mitochondria, a well-developed secretory apparatus, and numerous endosecretory granules). Such findings have been confirmed by light microscopy using randomized cell counting, with a significant increase of transitional or oxyphil cells compared with untreated HPC cells (preparation I: 63 ± 6% versus 19 ± 4%; p < 0.05; preparation II: 78 ± 6% versus 11 ± 7%, p < 0.01). Finally, HPC cells treated with 0.1 and 1.0 mM AZT presented a typical apoptotic morphology, with reduced cell volume, condensation of chromatin, and fragmentation of the nucleus. Typical apoptotic bodies were also observed (Fig. 3D). Similar observations were obtained in the two HPC cell preparations (I and II). Conversely, in the same experimental conditions, 100 μM AZT did not seem to induce any significant modifications in cells obtained from human adenomatous tissues (preparations III-V; Fig. 3D1).
Inhibition of HPC cell mitotic activity and proliferation by AZT
Mitotic activity of HPC cells was evaluated by [3H]thymidine uptake. AZT in a dose range from 0.1 μM to 1.0 mM inhibited [3H]thymidine uptake in a dose-dependent fashion (Fig. 4). No effects of AZT on DNA synthesis were observed on adenomatous human parathyroid cells (preparations III and IV; Fig. 4).
The effect of AZT on DNA synthesis was confirmed by the evaluation of HPC cell proliferation measured by cell counting. Human parathyroid cultured cells were incubated for 96 h with AZT in a range of concentrations from 0.1 μM to 1.0 mM. A dose-dependent inhibition of cell growth was observed in the two HPC cell preparations (I and II) from a concentration of 10 mM AZT, whereas no effect was evident on preparation III of human parathyroid adenomatous cells (Fig. 5).
In vitro induction of apoptosis in HPC cells by AZT treatment
HPC cells (preparations I and II) were cultured for 48 h in 0.1 μM to 1 mM AZT concentrations, and apoptosis was measured by annexin V flow cytometry. AZT induced apoptosis in a dose-dependent manner in both HPC cell preparations (Fig. 6). AZT did not increase apoptosis in human parathyroid adenomatous cells (preparations III-V; data not shown).
Intracellular AZT-MP levels correlate with sensitivity to AZT-mediated apoptosis and growth arrest
In AZT-sensitive HPC cells (preparations I and II), a markedly higher level of AZT-MP was detected than in resistant cell preparations (III-V; Fig. 7). This suggests that AZT-MP may preferentially accumulate in HPC cells sensitive to AZT-mediated apoptosis and growth arrest.
Parathyroid carcinoma represents an uncommon case of primary hyperparathyroidism.(14,15) Unfortunately, this rare disorder does not show any clarity to unequivocally address both clinical and pathological differential diagnosis with parathyroid adenoma or hyperplasia. The optimal treatment consists of an en bloc tumor resection with ipsilateral thyroid lobectomy when the diagnosis is suspected and until it is proven otherwise. Moreover, reoperation in patients with localized parathyroid carcinoma is recommended because it relieves symptoms of hypercalcemia, and it normalizes serum calcium and PTH levels in most patients. Patients who have persistent or recurrent parathyroid carcinoma should have localizing studies to identify loco-regional or distant tumor sites.(28) For patients with unresectable parathyroid carcinoma, a protocol-based treatment with chemotherapy and external radiotherapy should be considered.(29) Unfortunately, late diagnosis at a metastatic stage of this malignancy is not infrequent, and treatment with nonsurgical anticancer therapies is doubtful.(30) For all these reasons, the search for specific molecular markers to improve the precocious diagnosis of parathyroid tumors has had a great impact in the past decade.(31–36)
In a previous study, we observed increased telomerase activity in primary cultures of HPC cells compared with benign parathyroid lesions,(16) as indirectly confirmed by others,(17) suggesting telomerase activity as an additional molecular marker for parathyroid carcinoma diagnosis. Progressive shortening of telomeres constitutes a molecular mechanism by which normal cells avoid uncontrolled proliferation. Telomerase is present in nearly all immortal cell lines, germ-line cells, stem cells, and 90% of human tumors, but seldom in normal somatic cells.(23) Telomerase is a ribonucleic DNA polymerase that synthesizes telomeric repeats de novo and is involved in multiple cellular processes, including cell differentiation, proliferation, inhibition of apoptosis, tumorigenesis, and possibly DNA repair and drug resistance.(37–42) Most human cancers exhibit an upregulated or reactivated telomerase activity.(21) The selective expression of telomerase in tumor cells makes telomerase an attractive therapeutic target.
AZT is a thymidine analog, originally developed as an antineoplastic agent with biologic activity in phase I and II clinical trials in patients with solid tumors.(4–8) Acting as a false substitute for viral reverse transcriptase, AZT has been recently used almost exclusively as an antiretroviral agent.(9,43–45) AZT has multiple pharmacological actions, including inhibition of human telomerase reverse transcriptase component.(46) It has been established that AZT, alone or in combination with other anticancer drugs, is able to decrease the in vitro cell growth of several neoplastic cell lines.(2,3,9–13) This was further confirmed by in vivo clinical data both in primary and metastatic tumors.(2,3,47–54)
In this study, we investigated the possible mechanism of action of AZT by in vitro examination of several HPC cellular parameters, including morphology proliferation, apoptosis, differentiation, telomerase activity, and AZT metabolism. Human parathyroid cells derived from benign lesions served as controls. This study indicates that in vitro exposure of HPC cells to AZT resulted in inhibition of telomerase and intracellular accumulation of AZT-MP. Our data showed a correlation between intracellular levels of the metabolite AZT-MP, telomerase inhibition, and inhibition of HPC cell proliferation. We also found that AZT induced an apoptotic rather than a necrotic type of cellular death, as previously shown in Herpes virus-associated lymphomas.(11) Anticancer agents that induce tumor apoptosis would also likely lessen patient morbidity from complications of tumor lysis. The AZT effect was not observed in telomerase-negative human benign parathyroid cells. Collectively, these results support the hypothesis that targeting of telomerase activity produces antiproliferative effect in telomerase-positive HPC cells.
An interesting and unexpected finding was the AZT-induced morphological changes of HPC cells, which acquire a quiescent and less functioning phenotype, with no acute effects of AZT on PTH release. HPC cells treated with AZT showed features of oxyphil cells, characterized by a moderately developed secretory apparatus and rare endosecretory granules. Additional studies are needed to determine the mechanisms by which AZT induces ultrastructural changes of HPC cells and the potential conditions of this phenomenon with AZT pharmacological actions (i.e., inhibition of reverse transcriptase, human telomerase reverse transcriptase component, integrase, DNA polymerase γ, and thymidine kinase).(44–46)
Although further in vitro and in vivo evidence is needed, these results preliminarily indicate that AZT can be proposed as a potential antiparathyroid cancer agent. The tumor selectivity of this therapeutic approach is suggested by the finding that AZT is inactive in benign human parathyroid hyperfunctioning cells. To confirm these findings, more observations are necessary. However, if these observations are valid, they can be translated into clinical trials that use AZT, alone or in combination with other chemotherapeutic agents. This is extremely needed for a neoplasia that shows lack of response to classical anticancer treatments. Recently, molecules able to mimic the calcium effect, named calcium-mimetics or calcimimetics, activated the calcium-sensing receptor (CaSR) present on the cell surface of parathyroid cells and inhibited parathyroid function. Specifically, class II calcimimetics, NPS R568 and AMG 073 (cinacalcet HCl), have been successfully used in normalizing serum calcium levels in patients with parathyroid carcinoma.(55,56) In these patients, cinacalcet HCl was effective in reducing serum calcium levels, with serum calcium reductions maintained up to 18 months. Thus, cinacalcet HCl could potentially be an effective therapy for the treatment of hypercalcemia in patients with parathyroid carcinoma or recurrent primary HPT after parathyroidectomy (PTX),(56) associated with a subjective improvement in symptoms. Although the effects on tumor burden are still unknown, cinacalcet HCl seems to significantly ameliorate states of severe PTH-dependent hypercalcemia.(57) Undoubtedly, combinations of drugs such as AZT and calcimimetics, respectively acting on inhibition of parathyroid cells proliferation rate and PTH secretion, may offer an important opportunity to reduce the severity of both biological and clinical progression of parathyroid carcinoma.
This study was supported by A.I.R.C. 2000 and Fondazione Ente Cassa di Risparmio di Firenze (MLB).
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