Glioblastoma multiforme (GBM) is the most aggressive brain cancer. In this tumor, angiogenesis is so prominent to represent a landmark for histological diagnosis.1 Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, is expressed by endothelial cells (ECs) of the tumor vasculature with increasing incidence and relative quantity during the progression of astrocytic tumors toward malignancy. We first observed telomerase involvement in the angiogenesis of astrocytic tumors on human tissue sections.2 Expression of hTERT by vascular ECs is a phenomenon strictly related to tumor environment, since it is absent in the ECs of nonneoplastic granulation tissues as well as in normal brain.2 Moreover expression of hTERT is induced in ECs at an early stage of tumor progression and it is positively correlated with the histological grade of the tumor. Using in vitro models, we demonstrated that GBM is able to activate hTERT transcription as well as telomerase activity in human umbilical vascular endothelial cells (HUVECs) and that such effects are likely mediated by diffusible factor/s.3 Taken together, our data suggest that telomerase reactivation might improve the replicative potential of ECs, and that this phenomenon might be induced by the tumor itself. Telomerase expressing ECs are supposed to exhibit survival advantages, i.e. resisting the apoptotic cell death triggered by replication under the cell stressing conditions of the tumor environment.4 Here, we inhibited telomerase activity in human ECs, either by RNA interference (RNAi) targeting hTERT or by a dominant negative (DN) allele of hTERT, and demonstrated that telomerase is essential for the survival of ECs in a controlled bioassay of GBM angiogenesis. This result may have clinical impact for developing adjuvant therapies in highly angiogenetic malignancies.
Tumor angiogenesis is a complex process that involves a series of interactions between tumor cells and endothelial cells (ECs). In vitro, glioblastoma multiforme (GBM) cells are known to induce an increase in proliferation, migration and tube formation by the ECs. We have previously shown that in human GBM specimens the proliferating ECs of the tumor vasculature express the catalytic component of telomerase, hTERT, and that telomerase can be upregulated in human ECs by exposing these cells to GBM in vitro. Here, we developed a controlled in vivo assay of tumor angiogenesis in which primary human umbilical vascular endothelial cells (HUVECs) were subcutaneously grafted with or without human GBM cells in immunocompromised mice as Matrigel implants. We found that primary HUVECs did not survive in Matrigel implants, and that telomerase upregulation had little effect on HUVEC survival. In the presence of GBM cells, however, the grafted HUVECs not only survived in Matrigel implants but developed tubule structures that integrated with murine microvessels. Telomerase upregulation in HUVECs enhanced such effect. More importantly, inhibition of telomerase in HUVECs completely abolished tubule formation and greatly reduced survival of these cells in the tumor xenografts. Our data demonstrate that telomerase upregulation by the ECs is a key requisite for GBM tumor angiogenesis. © 2007 Wiley-Liss, Inc.
Material and methods
TB10 cell line was established in. our laboratory from a human GBM tumor.5 TB10 was grown in DMEM (high glucose, Invitrogen Italia, Milano, Italy) supplemented with 10% heat-inactivated fetal bovine serum. Immunohistochemistry for glial fibrillary acid protein (GFAP) was routinely performed to check maintenance of the glial phenotype. The cells were regularly tested for mycoplasma contamination. HUVECs (Bio-Wittaker, Walkersville, MD) were maintained in complete endothelial cell growth medium (EGM-2; Bio-Wittaker) containing endothelial cell basal medium (EBM-2; Bio-Wittaker) supplemented with endothelial cell Bullet kit (Bio-Wittaker; 2% FBS, hEGF-2, hVEGF, R3-IGF-1, ascorbic acid, hydrocortisone, heparin, gentamicin and amphotericin-B). Cells were grown at 37°C in a humidified atmosphere of 5% CO2–95% air. HUVECs used for the experiments were between passage 2 and 5. Hypoxic culturing conditions were achieved by Modular Incubation Chamber MIC-101 (Billups-Rothenberg, Del Mar-CA).
For immunostaining of cultured HUVECs and TB10, cells were plated onto poly-L-ornithine coated glass coverslips in serum-free medium. Cells were then fixed with 4% paraformaldehyde and stained with antibody directed against CD31 (Dako, Glostrup, Denmark), TERT protein (Novocastra Laboratories, Newcastle upon Tyne, UK), GFAP (Dako, Glostrup, Denmark) and GFP (BD Biosciences Clontech, Palo Alto, CA). Appropriate secondary antibodies (goat anti mouse IgG affinity purified TRITC-conjugate; Chemicon; goat anti rabbit IgG FITC-conjugate; Chemicon, Billerica, MA) were used.
Production of viral stocks and cell infections
p-BABE-puro-hTERT6 and p-BABE-puro-DN-hTERT7 were kindly provided by Robert A. Weinberg (Whitehead Institute for Biomedical Research-Cambridge). Amphotropic retroviruses were generated in Phoenix packaging cells upon transfection of the retroviral vectors by Lipofectamine reagent (Invitrogen Italia), according to the manufacturer's instructions. Forty-eight hr posttransfection, the viral supernatant was used to infect HUVECs after addition of 2 μg/ml polybrene. Cells were infected twice for 6 hr and, after 48 hr, selected with puromycin. Resistant HUVECs overexpressing hTERT or alternatively DN-hTERT were designed HUVEC-TERT and HUVEC-DN, respectively. To target hTERT by RNAi, we used the siTERT1 sequence that we have previously characterized in its ability to downregulate hTERT expression and telomerase activity.8 From the original construct p-RETRO-SUPERsiTERT1,8 a fragment encompassing H1 promoter and siTERT1 sequence was subcloned in the XhoI site of the lentiviral vector pCCLsin.PPT.hPGK.GFPpre.9 This vector contains the GFP gene driven by the human phosphoglicerate kinase promoter (hPGK). Amphotropic lentivirus was generated as described.9 Briefly, pCCLsin.PPT.hPGK.GFPpre construct was cotransfected in HEK 293 packaging cells with pMDL, pRSV-REV and pVSV-G by Lipofectamine reagent. Forty-eight hr posttransfection, the viral supernatant was used to infect HUVECs after addition of 8μg/ml polybrene. Cells were infected twice for 6 hr. Infection efficiency was monitored through GFP expression. Typically, 1 cycle of infection is sufficient to obtain virtually 100% of transduced HUVECs. HUVEC stably transduced with siTERT1 sequence or alternatively with empty vector were designed as HUVEC-siTERT and HUVEC-GFP, respectively. As negative control of interference, we used the rat sequence for the VGF gene, a neuronal specific mRNA that is not expressed in ECs,8, 10 generating HUVEC-siVGF.
Real time RT-PCR
hTERT expression was measured by Real-Time quantitative RT-PCR, based on TaqMan methodology, using ABI PRISM 7900 Fast Real Time PCR System (Applied Biosystems, Foster-City, CA). To normalize the amount of total RNA present in each reaction, a housekeeping gene, TBP, was amplified, which is assumed to be constant in all samples. Primers and probes were purchased from Applied Biosystems, Assay-on-Demand Gene Expression Products, TERT assay ID Hs001162669_m1 and TBP ID Hs99999910_m1.
Growth rate of HUVEC-GFP, HUVEC-TERT, HUVEC-siTERT and HUVEC-DN was studied by CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega Italia, Milano, Italy). Briefly, 5,000 cells per well were plated in a 96-multiwell plate. The data were averaged from 4 wells and expressed as mean ± SD of cell numbers per well. After 48 hr treatment the assay was performed according to the manufacturer's instructions.
HUVEC-TERT and HUVEC-GFP were treated with 0.75 mM H2O2 for 3 hr. After 15 hr of recovery in fresh EGM2 medium, cells were harvested, washed in cold PBS and fixed with cold 70% ethanol for at least 1 hr. After removing the alcoholic fixative, cells were stained for the analysis of the DNA content in a solution containing 50 μg/ml propidium iodide (Sigma) and 75 KU/ml RNase (Sigma-Aldrich, St. Louis, MO) in PBS, for 30 min at room temperature in the dark. Cell death was evaluated as percentage of the cells in the sub-G1 region in each DNA histogram, which is indicative of apoptosis.
For the Annexin V assay, we used HUVEC and HUVEC-TERT with no GFP labeling. Cells were harvested, washed once in cold PBS and processed according to manufacturer's instructions (Annexin V-FITC Apoptosis Detection Kit, MBL International, Woburn, MA). Ten thousands events/sample were acquired using a FACScan cytofluorimeter (Becton Dickinson, CA) and the analysis was performed by means of the CellQuest software package (BD).
HUVEC and glioblastoma cells xenografting in immunodeficient mice
Four-week-old male nude mice (Harlan, Udine, Italy) were used as hosts for the in vivo models of angiogenesis. The experiments on animals were approved by the Ethical Committee of the Catholic University School of Medicine, Rome. HUVEC-GFP, HUVEC-TERT, HUVEC-siTERT, HUVEC-siVGF and HUVEC-DN were harvested, washed twice and resuspended in cold PBS at the concentration of 1 × 104/μl. Then, 100 μl of cells were mixed with 100 μl of Matrigel (BD Bioscience, Bedford, MA) on ice, and the mixture was implanted by subcutaneous injection using a 25-gauge needle. Only one injection was performed on a single mouse. In TB10-HUVEC cografts, 5 × 105 TB10 cells and 5 × 105 HUVEC cells were resuspended in 100 μl of cold PBS, and the suspension was mixed with an equal volume of cold Matrigel. The animals were kept under pathogen-free conditions in positive-pressure cabinets (Tecniplast Gazzada, Varese, Italy).
Histology, immunohistochemistry and fluorescence microscopy
The Matrigel implants were removed by 1–4 weeks after grafting. The mice were deeply anesthetized and transcardially perfused with 0.1 M PBS (pH 7.4), followed by 4% paraformaldehyde in 0.1 M PBS. The implants were surgically removed, stored in 30% sucrose buffer overnight at 4°C and either embedded in paraffin or cryotomed. In paraffinized sections (5-μm thick), a previous step of heat-induced antigen retrieval technique by microwave oven processing (2 cycles of 5 min, 750 W) in citrate buffer was used. The sections were then incubated with anti-GFP (BD Biosciences Clontech, Palo Alto, CA). After incubation with the primary antibody, immunodetection was performed using the avidin biotin complex peroxidase method (ABC-px method) (LSAB-Dako, Golstrup, Denmark) using freshly made diaminobenzidine as a chromogen. Alternate sections were stained with H&E for morphological analysis. The material was studied under light field illumination and images were captured with a Leitz microscope equipped with a Nikon Coolpix 995 camera and connected to a PC. For fluorescence microscopy, cryotomed sections (20-μm thick) were collected in distilled water, mounted on slides and cover-slipped with Eukitt. Images were obtained with a Laser Scanning Confocal Microscope (IX81, Olympus, Melville, NY). For quantitative analyses, 10 separate fields were randomly selected per tissue section and the number of surviving GFP-positive HUVECs (20× fields) as well as the net-like tubule structures (10× fields) was counted and averaged. Representative images from each slide were acquired using the confocal microscope with fixed optical parameters, light intensity, filter settings and magnification. Acquired images were stored as TIFF files and evaluated using Adobe Photoshop 6.
Results are expressed as the mean ± standard deviation (SD). The significance of differences was assessed by the unpaired 2-tail Student's-t test. Significance was set at p < 0.05.
Preparation of engineered HUVEC cell populations
All the HUVEC populations used in the following experiments were induced to express GFP by lentiviral infection. Preliminary studies have shown that GFP expression in HUVECs does not alter their phenotype or culture behavior or telomerase expression (not shown). To test the effect of sustained telomerase expression in ECs, we generated via retroviral infection a population of HUVECs overexpressing hTERT. Reportedly, human ECs delay replicative senescence after introduction of hTERT4 due to reactivation of telomerase activity, which parallels hTERT overexpression. More specifically, HUVEC cells overexpressing an ectopic hTERT maintain their angiogenic potential in vitro and do not show neoplastic features because their growth is contact-inhibited, serum-dependent and anchorage-dependent. Notably, HUVEC-TERT lines maintain normal p53-dependent checkpoint control, inducing expression of p21 (Cip1/Waf1) in response to DNA damage.11 Since we have previously observed that HUVECs reactivate telomerase in the presence of human GBM cell lines,3 we generated HUVECs unable to enhance telomerase activity upon exposure to GBM. To this purpose, HUVECs were infected with a lentiviral construct which allows stable transduction of short hairpin RNAs (shRNAs) that are processed to siRNAs in mammalian cells.12 The si1 oligonucleotide encoding for shRNA complementary to hTERT was chosen based on its efficiency in inhibiting hTERT expression as well as telomerase activity in GBM cells.8 The resulting cell population was named HUVEC-siTERT. As control of RNAi, we generated a cell line, HUVEC-siVGF, obtained by lentiviral infection of a shRNA targeting the rat sequence of VGF gene, a neuronal specific mRNA that is not expressed in ECs (see Material and methods section). To test the efficiency of si1 sequence to downregulate hTERT in the HUVEC cell system, we performed Real Time RT-PCR on HUVEC-TERT transduced with si1 construct (HUVEC-TERT-si1), observing a 5-fold downregulation of hTERT with respect to HUVEC-TERT, demonstrating that the si1 sequence is efficient in inhibiting hTERT in HUVECs even when hTERT is abundantly expressed (Fig. 1). As expected, shRNA targeting VGF has no effect on hTERT mRNA expression (not shown).
Finally, HUVECs were infected with the retroviral construct p-BABE-puro-DN-hTERT, which codifies a catalytically inactive form of hTERT,7 which acts as a DN allele, generating HUVEC-DN.
Growth and morphological features of engineered HUVECs in vitro
The main consequence of hTERT gene transduction was that HUVEC-TERT showed an extended lifespan in vitro. We passaged HUVEC-TERT up to 50 population doublings (PDs), and we did not observe any change in their doubling time. As expected, both HUVEC with a canonical hTERT expression (HUVEC-GFP) and telomerase-inhibited-HUVECs (HUVEC-siTERT and HUVEC-DN) became senescent, flattened and underwent into growth arrest within a few passages. To address if telomerized HUVECs have any proliferative advantage with respect to primary or to telomerase-inhibited HUVECs, we evaluated the proliferation rate of HUVEC-TERT and of early passage HUVEC-GFP, HUVEC-siTERT and HUVEC-DN, either under standard culture conditions or under cell stressing conditions. To mimic the tumor microenvironment conditions, we conducted the experiment in low serum (0.5%), in glucose deprivation - adding to cell medium 1 or 5 mM deoxyglucose (DG) - or under hypoxic conditions, in hypoxic chamber (see Material and methods section). After 48 hr treatment, cells were analyzed by CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega). HUVEC-TERT exhibited a slight but statistically significant increase in proliferation rate with respect to control (p < 0.01, Student's t-test), except under hypoxic conditions, where all the groups displayed the same growth rate (Fig. 2a).
It has been reported that hTERT overexpression confers to the ECs an augmented resistance to apoptotic stimuli,4 we therefore treated control and telomerized HUVECs with hydrogen peroxide (H2O2). H2O2 is a well characterized mediator of oxidative stress which is known to generate a variety of different types of oxidative DNA lesions, including base modifications, frameshift mutations and DNA strand breaks.13 FACS analysis revealed a protection effect from apoptosis in HUVEC-TERT with respect to the other experimental groups, which displayed a homogeneous percentage of cells in sub-G1 in response to H2O2 treatment (Fig. 2b). An increased resistance to apoptotic stimuli by HUVEC-TERT was confirmed by Annexin V staining (Fig. 2c).
Although HUVEC-TERT showed an extended life-span in vitro and were more resistant to death than HUVEC-GFP, they did not exhibit features of transformed cells since they neither could be cloned nor were tumorigenic in immunodeficient mice (see later). Morphologically, HUVEC-TERT, HUVEC-siTERT and HUVEC-DN were identical to HUVEC-GFP, with characteristic cell extensions such as lamellipodia and pseudopodia, and expressed the endothelial specific antigen CD31 (Fig. 3). Immunostaining with anti-TERT antibody revealed that the low-passage subconfluent HUVEC-GFP had either a weak positive reaction, which stained diffusely the cell nucleus, or discrete granular staining outlining the nucleoli. In the HUVEC-siTERT, the degree of TERT immunostaining was similar to that of HUVEC-GFP, however, about 20% of the cells did not stain at all (Fig. 3). Conversely, HUVEC-TERT and HUVEC-DN showed an intense immunoreaction that diffusely stained cell nucleus obscuring any nucleolar detail (Fig. 3). The strong immunoreaction for TERT in DN-HUVEC is due to the accumulation of catalytically inactive hTERT which was recognized by the anti-TERT antibody.
hTERT inhibition abolishes the angiogenic behavior of HUVECs in GBM xenografts
Subcutaneous grafting of human ECs as Matrigel assay in immunosuppressed mice has previously been used to investigate survival of these cells under unfavorable in vivo conditions. For example, it has been demonstrated that primary human dermal microvascular ECs show poor survival and tubule formation when subcutaneously implanted in immunosuppressed mice, while their telomerized counterparts survive and form microvascular structures.14 Therefore, we used this model to test the hypothesis whether the presence of GBM tumor cells might affect the survival and the angiogenic behavior of grafted HUVECs through mechanisms that involve telomerase activation in these cells. In our bioassay of tumor angiogenesis, HUVECs with different degrees of telomerase expression and human GBM cells were cografted as Matrigel implants into the subcutaneous tissue of athymic nude mice. On histological examination, clusters of GBM tumor cells and neoformed vessels were found in Matrigel implants examined by 1 week after grafting (n, 5; Fig. 4, panel a). Immunostaining with anti-GFP and direct fluorescence microscopy revealed that HUVEC-GFP not only survived in the implant but even contributed to new vessel formation. At 2 and 4 weeks after grafting, the tumor xenografts (n, 12) were supplied by murine-human chimeric microvessels which were viable to circulating erythrocytes (Fig. 4, panel b). In experiments where HUVEC-TERT were used (n, 13), the overall picture was qualitatively similar to that of HUVEC-GFP (n, 12), although the number both of surviving cells and of tubule structures was significantly higher (p < 0.0001, t-test) (Fig. 5). Conversely, Matrigel implants containing HUVEC-siTERT (n, 16) or HUVEC-DN (n, 8) appeared completely devoid of GFP-labeled net-like structures, with only a few cells surviving (Fig. 5a). The total number of GFP-positive HUVECs was significantly lower in specimens containing either HUVEC-siTERT or HUVEC-DN relative to control HUVECs (HUVEC-GFP or HUVEC-siVGF) (p < 0.0001, t-test) (Fig. 5b). These data demonstrate that telomerase inhibition is sufficient to suppress the angiogenic behavior of the HUVECs in the tumor environment.
In the Matrigel implants which did not contain GBM tumor cells (n, 12), only a few HUVEC-GFP and HUVEC-TERT survived in the grafts (Figs. 5a, right panel and 5b), while both HUVEC-siTERT and HUVEC-DN did not (Fig. 5). Surviving HUVEC-TERT were interspersed within the Matrigel matrix as single cells without forming net-like structures. These findings demonstrate that primary HUVECs do not survive in Matrigel implant xenografts, and that hTERT upregulation, though increasing to some extent HUVEC survival, is not sufficient for these cells to elicit their contribution to angiogenesis.
We have previously shown that in human GBM the proliferating ECs of the tumor vasculature overexpress hTERT, and that hTERT expression by the ECs of the tumor vessels heralds the malignant progression of astrocytic brain tumors.2 A mechanism was postulated where GBM-derived factor/s would induce hTERT expression in the ECs of newly formed vessels. Increased transcription of hTERT may be a by-product, with no functional role, of the activation in ECs of a transcriptional program in response to angiogenic stimuli released by tumor cells. We have previously shown that ECs of astrocytoma tumors express high levels of cMyc.2 Since this oncogene is an established positive regulator of hTERT transcription15, 16 it is possible that hTERT expression is consequently upregulated without a specific role. Conversely hTERT expression, and the consequent upregulation of telomerase activity, may be important for tumor angiogenesis. Accordingly, it has been demonstrated that tumor vascularization is impaired in telomerase-negative mice with short telomeres.17 In the attempt to address this point we generated both HUVEC where telomerase was upregulated, and HUVEC where telomerase upregulation was prevented either by RNAi targeting hTERT or by the overexpression of a DN allele of hTERT. Then, we analyzed survival and tubule formation of these HUVEC strains in subcutaneous xenografts containing human GBM cells. This model should simulate the interactions between GBM tumor cells and human ECs. We found that hTERT expression in HUVECs is essential for their ability to survive and form tubule structures in the tumor environment. The angiogenic potential of HUVECs was strongly influenced by hTERT expression in this model. When hTERT is overexpressed, HUVECs survive in the implant and even form chimeric murine-human microvessels. Conversely, when hTERT reactivation is prevented, no tubule formation is present in the xenograft and only a few HUVECs survive. Since the same finding holds true upon overexpression of DN-TERT, we conclude that telomerase activity and not simply hTERT expression is required for HUVECs survival in the cograft with GBM. It is important to note that the angiogenic behavior of engrafted HUVECs depends on the presence of GBM cells, in fact telomerized HUVECs show poor survival when subcutaneously grafted in nude mice as single culture. Thus, telomerase overexpression is a necessary condition but not a sufficient one to allow HUVEC survival and formation of vascular structures in subcutaneous Matrigel implants.
The issue whether hTERT overexpression is a condition sufficient per se to allow proliferation and ECs organization in net-like structures is controversial. Our study demonstrates that hTERT overexpression might allow only a very limited survival, which is not sufficient to elicit any angiogenic behavior of HUVECs in the Matrigel implant. Conversely, Yang et al. showed that retroviral-mediated transduction of hTERT in HDMECs resulted in cell lines that form microvascular structures when subcutaneously implanted in SCID mice, with functional murine-human vessel anastomoses.14 In agreement with our data, however, Freedman and Folkman showed that telomerized HUVECs, though capable of extended proliferation in vitro, do not grow or survive when implanted subcutaneously in immunocompromised mice.11 The orthotopic model used for HDMEC xenografting versus the heterotopic one used for HUVECs might be influential in explaining such discrepant results. Beside this, the key aspect of the present study is that the proangiogenic effect of GBM-related factor/s is nearly abolished when we prevent the ability of HUVECs to reactivate hTERT.
Telomerase has also been demonstrated to be an important inducer of angiogenic phenomena in nonneoplastic contexts. For example, Murasawa et al. demonstrated that telomerase activity contributes to endothelial progenitor cells angiogenic properties: mitogenic activity, migratory activity and cell survival resulted significantly enhanced by hTERT overexpression.18 In our model system we found no evidence of HUVECs proliferation within the graft, in fact the number of HUVECs in the xenograft at any survival time was smaller than the number of grafted cells. Thus it is unlikely that the higher number of surviving telomerized HUVEC is due to an increase in replication potential provided by telomerase activity. We favor the hypothesis that telomerase expression increases resistance of HUVECs to apoptotic stimuli. Several reports describe increased cell death in normal and transformed cells upon telomerase inhibition independently of telomere shortening19–22 as well as protection from cellular stresses of nontransformed cells upon telomerase overexpression.23 Recently it was shown that inhibition of telomerase expression in normal human fibroblasts abrogates the cellular response to DNA double strand breaks, impairs the DNA damage response and decreases DNA repair.24 In addition, low level of telomere dysfunctions in presenescent normal human mammary epithelial cells was shown to lead to a subthreshold activation of p53, a phenomenon abrogated by ectopic expression of telomerase.25 p53 appeared to be capable of integrating signals originating from telomere dysfunctions and from growth factor deprivation, thus inducing cell cycle arrest. Thus signals from telomere dysfunctions can be similarly integrated with different forms of cellular stresses leading to apoptosis. Our finding showing that telomerized HUVEC are more resistant to oxidative stress induced by a pulse of H2O2 is compatible with this hypothesis.
In conclusion, our study demonstrates that the selective inhibition of telomerase in the ECs of GBM xenografts abolishes the angiogenic behavior of these cells in the tumor environment. Other than a direct effect on GBM tumor cells,8 antitelomerase compounds might hinder tumor growth through an antiangiogenetic mechanism directed toward the ECs of the tumor vasculature. Compared to the poorly vascularized core region of the tumor, the ECs represent a much more attractive target for drugs delivered into the blood stream.
M.L.F. is supported by Filas. The authors thank Prof. Robert Weinberg (Whitehead Institute for Biomedical Research-Cambridge) for the hTERT dominant negative construct, and Mrs. Adele De Santis for helpful assistance in editing of figures.