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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Int J Cancer. Author manuscript; available in PMC 2010 July 1.
Published in final edited form as:
PMCID: PMC2782444
NIHMSID: NIHMS143590

The antiretroviral nucleoside analogue Abacavir reduces cell growth and promotes differentiation of human medulloblastoma cells

Abstract

Abacavir is one of the most efficacious nucleoside analogues, with a well-characterized inhibitory activity on reverse transcriptase enzymes of retroviral origin, and has been clinically approved for the treatment of AIDS. Recently, Abacavir has been shown to inhibit also the human telomerase activity. Telomerase activity seems to be required in essentially all tumours for the immortalization of a subset of cells, including cancer stem cells. In fact, many cancer cells are dependent on telomerase for their continued replication and therefore telomerase is an attractive target for cancer therapy. Telomerase expression is upregulated in primary primitive neuroectodermal tumours and in the majority of medulloblastomas suggesting that its activation is associated with the development of these diseases. Therefore, we decided to test Abacavir activity on human medulloblastoma cell lines with high telomerase activity. We report that exposure to Abacavir induces a dose-dependent decrease in the proliferation rate of medulloblastoma cells. This is associated with a cell accumulation in the G2/M phase of the cell cycle in the Daoy cell line, and with increased cell death in the D283-MED cell line, and is likely to be dependent on the inhibition of telomerase activity. Interestingly, both cell lines showed features of senescence after Abacavir treatment. Moreover, following Abacavir exposure we detected, by immunofluorescence staining, increased protein expression of the glial marker glial fibrillary acidic protein (GFAP) and the neuronal marker synaptophysin (SYN) in both medulloblastoma cell lines. In conclusion, our results suggest that Abacavir reduces proliferation and induces differentiation of human medulloblastoma cells through the downregulation of telomerase activity. Thus, using Abacavir, alone or in combination with current therapies, might be an effective therapeutic strategy for the treatment of medulloblastoma.

Keywords: Abacavir, differentiation, medulloblastoma, reverse transcriptase, telomerase, senescence

At every round of cell division human chromosomes lose a certain amount of DNA at the telomeres, which is the basis of the limited replicative lifespan of normal cells1. Telomerase is a ribonucleoprotein enzyme consisting of three components, human telomerase RNA, telomerase-associated protein and human telomerase reverse transcriptase (hTERT). This enzyme utilizes its own RNA as template to synthetize telomeric DNA that, together with telomere-binding proteins, confers stability to chromosomes counteracting the telomere-dependent pathways of cell mortality.1 Importantly, telomerase activity is detectable in over 80% of human tumour samples in vivo, including most of the common and therapeutically intractable types, and some studies have suggested that cancer stem or stem-like cells are also telomerase-positive2. Conversely, telomerase is not expressed in normal cells before birth and is expressed only transiently or at low levels in proliferative tissues after birth3. Thus, although telomerase itself is not considered an oncogene, its repression and tight regulation in humans function as tumour suppressor mechanisms, and it's now well-established that telomerase plays an important role in cellular immortalization and tumorigenesis4. The reactivation of telomerase in cancer cells stabilizes telomere length, thereby counteracting the cell division-related telomere erosion and providing unlimited proliferative capacity to malignant cells. This makes telomerase an attractive target for cancer therapy5. The key advantages of targeting telomerase in comparison with most other cancer targets are its wide expression in tumours and its specificity for cancer cells, including putative cancer stem cells1,6. No other tumour-associated gene is as widely expressed in cancer1,6. Furthermore, the low or transient expression of telomerase in normal tissues and the longer telomeres in normal cells, compared with those in tumours cells, reduce the probability of toxicity, associated to telomerase-based drugs, in normal cells suggesting that this kind of therapy could have a broad therapeutic window1. Moreover, as late stage and recurrent human tumours are characterized by immortal cells that have reactivated telomerase, telomerase antagonists may be useful also when traditional anti-tumour therapies, which are generally more effective against early stage cancer, have failed7. Thus, anti-telomerase strategies promise to be a novel anti-cancer approach that might be effective also against disseminated advanced tumours.

Reverse transcriptase (RT) inhibitors belong to a family of 19 compounds used for the treatment of human immunodeficiency virus (HIV) infections8. They include the nucleoside RT inhibitors (NRTIs), the nucleotide RT inhibitors (NtRTIs) and the non-nucleoside RT inhibitors (NNRTIs)8,9. After intracellular phosphorylation, NRTIs and NtRTIs function as competitive inhibitors of the normal substrates (either dATP, dGTP, dCTP or dTTP) and lead to the termination of chain elongation, thereby blocking DNA synthesis. Whereas NNRTIs inhibit RT activity by direct binding the hydrophobic pocket in the p66 subunit of RT enzyme, near the polymerase active site8,9.

Abacavir, (1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol succinate (ABC), also known as Ziagen, is a 2′-deoxyguanosine analogue, which has potent antiretroviral properties and therefore it has been approved for the clinical treatment of AIDS in adults and in children10. ABC is one of the most efficacious nucleoside analogues in the treatment of AIDS and it has a favourable safety profile and desirable pharmacokinetic characteristics, such as bioavailability11. In fact ABC is well and rapidly absorbed following oral administration and it has an absolute bioavailability of approximately 83%. ABC is methabolized, through a unique phosphorylation pathway, to a carbocyclic guanosine triphosphate, carbovir triphosphate (CBV-TP), which is a substrate for the HIV RT and causes DNA chain termination owing to the lack of 3′-OH12. According to Tendian et al.13 the active form of ABC, which is a substrate for the HIV RT, is also an inhibitor of human telomerase activity. In fact CBV-TP is incorporated into DNA at the telomeres by telomerase, thereby blocking the extension of the DNA chain. This study was supported by the observation of Yegorov et al.14 showing the inhibition of telomerase activity in mouse fibroblasts following exposure to CBV-TP, the active metabolite of ABC.

Telomerase expression is upregulated in 76% of primary primitive neuroectodermal tumours15, and in neuroblastoma the level of the enzyme correlates with high genetic instability and a poor clinical outcome1. More recently, Chang et al.16 showed that telomerase activity is present in the majority of medulloblastomas, suggesting that telomerase activation is associated with the development of these diseases. Despite improvements in the overall survival rate of patients with medulloblastoma, owing to the multimodality treatment that includes surgery, radiation and chemotherapy, some patients will have recurrent or progressive disease17,18,19. Unfortunately, attempts to further reduce the morbidity and mortality associated with medulloblastoma are limited by the toxicity of conventional treatments and the infiltrative nature of the disease17. Thus, there is an urgent need to develop innovative therapeutic approaches that can improve survival and reduce toxicity. Therefore, we decided to test ABC activity on two human medulloblastoma cell lines, Daoy and D283-MED, in which a high telomerase activity is documented15,16. We report that exposure to ABC is associated with decreased cell growth rate in medulloblastoma cells and this effect is accompanied by the promotion of differentiation.

Material and Methods

Cell cultures and Abacavir treatment

The human medulloblastoma cell lines Daoy and D283-MED were obtained from American Type Culture Collection (ATCC, Manassas, VA). Daoy cell line was established from a desmoplastic medulloblastoma from a 4-year-old male and grows as attached polygonal cells with a population doubling time of 30,5 h (He et al.20). D283-MED was established from the peritoneal metastasis of a 6-year-old male with medulloblastoma and grows as a mixed culture of both attached and suspension cells21. These cell lines were routinely cultured in DMEM (CellGro, Mediatech, Inc., Herndon, VA) supplemented with 10% foetal bovine serum (Atlanta Biological, Norcross, GA) at 37°C in a humidified atmosphere of 5% CO2 in air, according to the ATCC recommendations.

Abacavir was purified from commercially available Ziagen (GlaxoWellcome) by Dr Raffaele La Montagna and dissolved in DMEM to make a 1mg/ml stock solution. Cells were plated at the initial density of 700 × 103/100mm diameter plate and 24h later were exposed to the drug. Cells were harvested by trypsinization at different time points and cell viability was assessed using the trypan blue dye exclusion method in a Burker chamber (three counts per sample).

Cell cycle analysis

For cell cycle analysis, 1 × 106-aliquots of cells were harvested and washed twice with cold phosphate-buffered saline (PBS), fixed in 70% ethanol and stored at 4°C until the analysis. Following centrifugation at 1000rpm, the resulting cell pellet was incubated, in the dark, in 0,3ml of freshly prepared PBS containing 0,02mg/ml propidium iodide and 0,25mg/ml ribonuclease A (Sigma, Sigma-Aldrich). The DNA content of the cells was analyzed using a FACStar Plus flow-cytometer (Beckton-Dickinson) (10000 events/sample).

Telomerase activity assay

Telomerase activity was determined by the telomeric repeat amplification protocol (TRAP)22. To this purpose, cells were treated with 750μM of Abacavir for 1, 2, 3, 4 or 5 days and were washed once in PBS. Then the pellets (~1 × 105 cells) were suspended in 400μl of TRAPeze CHAPS lysis buffer (Chemicon International, Temecula, CA). The samples were incubated for 30 min on ice and then centrifugated for 30 min (14000 RPM, 4°C). The supernatant was collected, snap-frozen and stored at -80°C. Protein concentration of the extract was measured using the Quick Start Bradford Dye Reagent (Bio-Rad Laboratories, Inc, Hercules, CA). For each sample 4μg of proteins were incubated with 75mM Tris-HCL (pH8.8 at 25°C), 20mM (NH4)2SO4, 0.01% Tween 20, 200μM dNTPs, 100ng of TS primer (5′-AATCCGTCGAGCAGAGTT-3′) in a thermocycler for 31 min at 30°C for the generation of telomeric repeats. After heating at 94°C for 5 min and cooling at 72°C, 2.5 U of Taq DNA polymerase (Fermentas, Life Sciences), 100ng ACX return primer (5′-GCGCGG(CTTACC)3CTAACC-3′), 100ng of NT internal control primer (5′-ATCGCTTCTCGGCCTTTT-3′), and 0,02amol of TSNT internal control (5′-AATCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT-3′) were added to a total reaction volume of 50μl. Thirty PCR cycles (94°C for 40 s, 56°C for 55 s and 72°C for 55s) were performed and the PCR products were electrophoresed on a 10% polyacrylamide nondenaturing gel in TBE. Electrophoresis was carried out at 200V in TBE at room temperature until the bromophenol blue just ran off the gel. Then, the gel was analyzed following silver staining according to Dalla Torre CA et al.23 The appearance of a 6-nucleotide ladder starting at 50bp indicates the presence of telomerase activity. The TRAP assay included the amplification of an internal telomerase assay standard band (ITAS), a primer-dimer/PCR contamination control and a negative control from Daoy and D283-MED cell lines after heat inactivation (85°C for 10 min).

Immunofluorescence detection of glial and neuronal differentiation markers

After 96h of treatment with ABC, Daoy and D283-MED cells were fixed in 3.7% (v/v) formaldehyde/PBS and permeabilized with 90% methanol/PBS (v/v). Then, cells were incubated with 1μg/ml of monoclonal antibody against the glial marker GFAP or polyclonal antibody against the neuronal marker SYN (Santa Cruz Biotechnology, Inc.), washed and incubated with secondary FITC-conjugated antibodies (1/1000, Vector Laboratories, Burlingame, CA). The samples were mounted in Vectashield (Vector laboratories) containing 4,6-Diamidino-2-Phenylindole (DAPI) in order to visualize the nuclei, then analyzed by fluorescence microscopy.

Real Time RT-PCR

To analyze the expression levels of GFAP, SYN and hTERT mRNAs during the treatment of ABC, real-time RT-PCR was performed with Power SYBR green PCR master mix (Applied Biosystems, Foster city, CA) in Real-Time PCR Systems 7500 Fast (Applied Biosystems, Foster city, CA). Daoy and D283-MED cells were harvested after 4, 8, 12, 24, 48, 72 and 96 hours of treatment with Abacavir (750μM) and total RNAs were extracted with Nucleospin RNA II (Macherey-Nagel) in diethyl pyrocarbonate-treated water. cDNA was synthetized with M-MuLV Reverse Transcriptase RNase H- (Finnzymes, Finland) and 100ng random hexamers in a total volume of 20μl from 1μg of total RNA. For PCR the following primers were used: for GFAP (accession No.:NM_002055) forward 5′-TGGAAGCCGAGAACAACCT-3′, reverse 5′-CCTCCAGCGACTCAATCTTC-3′; for SYN (accession No.:NM_003179.1) forward 5′-GTGACCTCGGGACTCAACAC-3′, reverse 5′-AGCCTGTCTCCTTAAACACGAA-3′ and for hTERT (accession No.:NM_198253) forward 5′-CGGAAGAGTGTCTGGAGCAA-3′, reverse 5′-GGATGAAGCGGAGTCTGGA-3′. Gene expression level was normalized to the value of the internal standard HPRT1 (accession No.:NM_000194) forward 5′-AGCCAGACTTTGTTGGATTTG-3′, reverse 5′-TTTACTGGCGATGTCAATAAG-3′, and the differences in gene expression were calculated by the standard ΔΔCt method. All the reactions were performed twice in triplicate and the specificity was assessed by analysis of the dissociation curve generated from each reaction and electrophoretic run on a 2% agarose gel.

Senescence-associated β-galactosidase activity

SA-β-Gal activity was determinated using β-galactosidase staining. Cells were washed in PBS, fixed for 3-5 min (room temperature) in 3% formaldehyde, washed, and incubated at 37°C (in absence of CO2) with fresh SA-β-Gal stain solution: 1 mg/ml of 5-bromo-4-chloro-3-indolyl β-D-galactoside (X-Gal) (Promega), 40 mM citric acid, sodium phosphate, pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl2. Staining was evident in 12-16 hr.

Statistical analysis

Data were expressed as mean ± standard deviation (SD) of three independent experiments and statistical significance was evaluated using Student's t test. For each statistical analysis, the associated probability (p value) of <5% was considered significant. Statistical differences between controls and samples were indicated by * for P<0.05 or ** for P<0.01.

Results

Abacavir inhibits the growth of medulloblastoma cells in vitro

As a first step to test the effect of ABC in the human medulloblastoma cell lines Daoy and D283-MED, we analyzed the growth rate of cells exposed to different doses of the drug compared with non-exposed controls. We decided to start with the doses used for the non nucleosidic RT inhibitor nevirapine, which has demonstrated cytotoxicity in vitro in melanoma and prostate cancer24. As shown in Figure 1a, a dose-dependent effect of ABC on the cell growth rate of cells was observed, with the maximal growth suppression at the 550-750μM dose range, an intermediate response at 350μM, and no suppressive effect below the 250μM dose (data not shown), in the cell lines tested.

Figure 1Figure 1Figure 1
Proliferation of human medulloblastoma cell lines Daoy and D283-MED treated with Abacavir (ABC). (a) Cells were cultured with different doses of ABC (dashed lines) and in drug-free medium (solid line). The cell proliferation rate is indicated as the ratio ...

At the doses of 550 and 750μM of ABC we observed a massive decrease in cell density at early time points, which was accompanied by an evident change in morphology. Cells appeared much larger in size and grew as monolayer with evidence of neurite formations (Fig. 1b).

Next, we analyzed by flow cytometry the cell cycle of cells treated with the different doses of ABC, in order to test whether the exposure to the drug has also effects on the cell cycle. Figure 2 shows the cell distribution across the phases of cell cycle at different time points during ABC treatment. At 48 h of exposure to ABC, Daoy cells accumulated in the S and G2/M phases of cell cycle, in a dose-dependent manner. At the maximal dose of the drug we observed: 32.3% in S phase, 37.3% in G2/M phase for ABC-treated cells vs 21.5% and 21.8% respectively for untreated cells. Interestingly, at 72 h interval, we observed a complete shift of treated cells into G2/M phase: 26.9% in S phase, 37.8% in G2/M phase vs 16.8% in S phase and 19.0% in G2/M phase in control cells and, after 2 weeks of treatment, ABC induced cell death (Figure 2b). The cytotoxic effect was more pronounced in D283-MED. In fact, as evidenced by FACS analysis, cells with a sub G1 DNA content accumulated after 48h of treatment with all doses of ABC. A maximal effect was observed with 750μM dose of ABC in both cell lines (Figure 2b). Therefore, we focused our studies on the 750μM dose of ABC. To test the effect of ABC and its eventual cytotoxicity on normal cells, we used primary cultures of rat neurons. We did not observe morphological changes such as loss of neuronal processes, at the doses of 550 and 750μM, as evidenced by the photographs in figure 1c obtained before and after exposure to 750μM ABC. Given that neurons are non proliferative cells, we have also tested whether ABC has effects in the cell cycle of the immortalized neuronal cell line, HT22, from mouse hippocampus. No effect was observed in the cell cycle profile of ABC-treated cells compared to untreated cells, also at the higher dose tested (Fig. 2c).

Figure 2Figure 2Figure 2
Flow cytometric profiles of Daoy and D283-MED medulloblastoma cells and HT22 neuronal cell line before and following exposure to Abacavir (ABC). (a) FACS analysis was performed after 24, 48, 72, 96 h and 2 weeks following 750μM ABC treatment. ...

Abacavir induces the downregulation of telomerase activity in medulloblastoma cells

To assess the effect of ABC on telomerase activity, we performed a TRAP assay in both cell lines at 1, 2, 3, 4 and 5 days after treatment with 750μM of ABC. We found that the antiproliferative effect of ABC is accompanied by the inhibition of telomerase activity as evaluated by the decrease of the 6bp ladder beginning from 1 d of treatment (Fig. 3a). Interestingly, also the level of hTERT mRNA, encoding the catalytic subunit of telomerase, was downregulated as early as 1 d following ABC treatment, as shown by real-Time RT-PCR (Fig. 3b). This finding suggests that ABC might inhibit telomerase activity not only by acting as guanosine analogue in DNA synthesis at the telomeres, as previously reported13, but also through the downregulation of hTERT mRNA transcription.

Figure 3Figure 3
Abacavir (ABC) inhibits telomerase activity and downregulates hTERT mRNA. (a) Detection of telomerase activity in medulloblastoma cells by TRAP assay. The detection of 6bp ladders indicates the presence of telomerase activity. Daoy and D283-MED cells ...

Exposure to Abacavir promotes differentiation in medulloblastoma cells

According to Gao K. et al.25 telomerase is repressed during lineage stem cell maturation in embryonic development and during differentiation of various immortal cells,26,27,28 suggesting that differentiation is associated to the downregulation of telomerase activity. Therefore, we investigated whether ABC is able to influence also the process of differentiation in Daoy and D283-MED cells. Medulloblastoma cells express various markers of cell differentiation, such as those of the neuronal and glial lineage, but also of cartilage, muscle, fat and mesenchyme29,30. Furthermore, it was shown that adjuvant therapy can induce extensive or complete neuronal maturation in medulloblastoma in two medulloblastoma cases, suggesting that these tumour cells can differentiate31.

Following ABC exposure, we detected in both cell lines analyzed, by immunofluorescence staining, increased protein expression of SYN and GFAP, which is one of the intermediate filaments found in mature normal astrocytes and differentiated glioma cells. SYN and GFAP increase became apparent in cells after 3 days of exposure to 550 and 750μM of ABC, as indicated in figure 4a. Whereas, at lower doses (350μM), the drug causes only a marginal increase in GFAP and SYN protein levels after 1 week (data not shown). As expected, no or low levels of protein expression were found, instead, in untreated cells. Real-Time RT-PCR confirmed the pattern of increased gene expression of both GFAP and SYN in Daoy cells following 2 days of exposure and in D283-MED at earlier time of treatment (Fig. 4b). These data are consistent with the morphologic changes and the neurite formation described above and showed in figure 1b.

Figure 4Figure 4Figure 4
Abacavir (ABC) induces differentiation and cellular senescence in medulloblastoma cells. (a) Immunofluorescence staining for GFAP and SYN proteins in Daoy and D283-MED cells. Expression of GFAP and SYN protein was evident in cells treated with 750μM ...

Abacavir induces senescence in medulloblastoma cell lines

Telomerase inhibition gives rise in cells to a telomere-induced senescence that is triggered by the formation of critically short and dysfunctional telomeres27. Cellular senescence is a tumour suppressive mechanism that mediates the anticancer effects of many chemotherapies32. Interestingly, one of the morphologic changes that was observed in our ABC-treated cells is the flattening of cells, which is a typical morphologic change associated with cellular senescence32. By β-galactosidase staining, we confirmed that cellular senescence was induced in D283-MED and Daoy cells by ABC in a time- and dose-dependent manner beginning from 1 week of treatment. Both cell lines showed a substantial senescent phenotype also at the dose of 350μM as shown in figure 4c.

Discussion

Telomerase is a relatively specific cancer target as normal body cells express little or no telomerase for most of their lifespan and generally have longer telomeres compared with tumour cells1. ABC is a specific RT inhibitor that has been approved for clinical use in the treatment of AIDS and is reported to be well tolerated in patients33. Furthermore, it has been shown that ABC exerts an inhibitory effect on the telomerase activity. In this report, we showed that ABC inhibits cell growth of the human medulloblastoma cell lines Daoy and D283-MED and this effect is associated with the downregulation of telomerase activity. To date, two major therapeutic approaches against telomerase-positive tumour cells are in clinical trials. GRN163L, a direct telomerase inhibitor, is in trial for the treatment of chronic lymphocytic leukaemia, multiple myeloma, solid tumours and non-small cell lung cancer (NSCLC)34. Several therapeutic vaccines targeting hTERT are in, or have completed, trials for the treatment of leukaemia and renal, prostate, lung, skin, pancreatic and breast cancer1. Compared with other tested compounds targeting the telomerase, or telomerase therapeutic vaccines, which are currently in clinical trials, ABC has been used for many years for the treatment of AIDS10,35. Therefore, the prospect of using this drug in cancer therapy would have obvious advantages given its favourable safety profile and its epidemiological record of generally good tolerance to continued administration. Indeed, we did not observe cellular toxicity, following ABC treatment, in two normal neuronal cell types of murine origin.

Here, we observed that the ABC-dependent inhibition of cell proliferation in D283-MED cells is accompanied by a dramatic increase in cell death. This is in accordance to another study showing that morphogenetic protein-7 (BMP7) induces telomerase inhibition, telomere shortening, and cell death by a mechanism involving the repression of hTERT, in cervical cancer cells.36

Whereas, in the Daoy cell line, along with the decrease in cell proliferation, we observed a significant cell accumulation in the G2/M phase of the cell cycle, following exposure to ABC. ABC did neither cause significant cell death nor apoptosis as indicated by FACS measurement of cells with hypodiploid DNA content, suggesting that decreased cell proliferation rather reflects a progressive withdrawal from the cell cycle. Telomerase is a ribonucleoprotein complex that adds telomeric repeats onto the ends of chromosomes during the replicative phase of the cell cycle. In fact, the highest levels of telomerase activity are found in cells that are in the S-phase of the cell cycle, whereas cells in G2/M phase have little or no telomerase activity37. This is in accordance to other studies showing that the inhibition of telomerase activity is associated to a block in the G2/M phase of the cell cycle38. One of these studies showed that phorbol 12-myristate 13-acetate induced a cell cycle arrest in G2/M in asynchronously growing NSCLC cells and conferred morphological and biochemical features of senescence, including downregulation of telomerase activity39. Our result show that ABC treatment leads Daoy cells to arrest in G2/M whereas it induces cell death in D283-MED, likely through the inhibition of the telomerase. However, further studies to examine the molecules implicated in the regulation of the G2/M phase checkpoint and in the cell death programme are crucial to understand the molecular mechanism of ABC, and they are currently under investigation in our laboratory.

Consistent with the central role of telomere shortening in the replicative senescence programme39,32,40, ABC was also able to induce cellular senescence in both medulloblastoma cell lines. This is very interesting considering that senescence is a powerful tumour suppressive mechanism. But whether the senescent phenotype is a direct consequence of telomere erosion, or rather it is due to telomere length-independent changes remains to be established. In fact, it has been reported that following telomerase repression other mechanisms that are independent from telomere erosion can trigger a damage response and abrupt cell-cycle arrest or death1,41,42.

Moreover, the elevated expression of GFAP and SYN markers, both at mRNA and protein levels, along with the neurite formation observed in both cell lines following two weeks of treatment with ABC, suggested that a more differentiated phenotype had been induced. The induction of differentiation occurring in our experiments after ABC treatment is typical of others inhibitors of telomerase activity and is likely to be due to an altered pattern of gene expression43. According to Damm et al43, in fact, the drug-mediated telomerase inhibition in cancer cells has been reported to reduce proliferation and induce changes in gene expression, along with the appearance of senescence hallmarks. Our results are in accordance to another study, in which anticancer effects of aloe-emodin involve G2/M cell accumulation concomitant with the induction of differentiation44. In addition, retinoic acid derivatives showed induction of cell differentiation, apoptosis, and growth arrest in some medulloblastoma cells. In particular, the results demonstrated that cell growth arrest was the main mechanism by which RA inhibited cell proliferation in the medulloblastoma cell line Daoy, but not in the others (D283-MED and D341), in which it induces apoptosis.45 Therefore it is possible that ABC inhibits cell proliferation, by G2/M block or cell death depending on cell type, and induce differentiation through the inhibition of telomerase activity. Even if the molecular mechanism by which ABC inhibits proliferation and induce differentiation are still to be clarified, the finding that ABC can induce the expression of differentiation markers in medulloblastoma cells suggests that it may attenuate the malignant and aggressive phenotype of transformed cells slowing the progression of disease. Molecular oncology studies, in fact, have shown that the genes regulating the differentiation process are not lost in malignant cells but the signals regulating cell proliferation, apoptosis and differentiation are defective. Thus, it is important to find agents that are able to restore the regulating machinery that will induce tumour cells to differentiate and revert their malignant phenotype. Interestingly, growth inhibition and increased sensitivity to anticancer drugs were observed in Daoy cells transfected with GFAP46. Such differentiation could also make tumour cells more responsive to normal growth regulatory signals or more sensitive to chemotherapy and other differentiation agents47. For instance, the remarkable success of all-trans-retinoic acid in producing complete remission in patients with acute promyelocytic leukemia has attracted interest in differentiation as an alternative form of cancer chemotherapy48. Treatment with retinoids can also induce differentiation in astrocytes, together with telomerase activity downregulation and increase of cell sensitivity to taxol49.

Another hurdle in the treatment of medulloblastoma is that the blood-brain barrier restricts the entry of hydrophilic and large lipophilic compounds into the brain17,35. The lipophilic nature of ABC, which enables it to pass through the blood-brain barrier more easily50, represents yet another advantage of its possible use for the treatment of medulloblastoma. Moreover, given its good tolerability, ABC treatment could be associated to current therapies used for medulloblastoma, including chemotherapeutic alkylating agent, such as cisplatinum and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, or mitotic inhibitors, such as vincristine17,51.

In conclusion, we suggest that the use of ABC, which is currently used in the treatment of AIDS, could be an effective therapeutic strategy, alone or in combination with current therapies, for the treatment of telomerase expressing tumours, such as human medulloblastomas.

Acknowledgments

This study was supported by NIH grants to A.G. and by the Sbarro Health Research Organization.

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