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Telomerase is a key oncogenic enzyme, and a number of novel telomerase inhibitors are currently under development. Because inhibition can be achieved either at the protein or at the enzymatic activity level, independent measurements of these parameters are important in the development of effective therapeutic agents. In the current study, we have developed a set of functional magnetic nanosensors capable of measuring the concentration of telomerase, as well as its enzymatic activity in parallel. The method is based on a magnetic relaxation switch assay, which can be performed in crude tissue samples and is fast and extremely sensitive. Using this method, we were able to detect different amounts of telomerase protein and activity in various cancer and normal cell lines. Furthermore, we were able to study the effect of phosphorylation on telomerase activity. This system not only could provide a rapid assay for the evaluation of antitelomerase therapies but could also be implemented to the study of other cancer markers.
Human telomerase (hTERT) is a ribonucleoprotein reverse transcriptase that catalyzes the addition of telomeric repeat units (TTAGGG) to the telomere's end of each chromosome [1,2]. Most somatic cells do not contain detectable levels of active telomerase; thus, their telomeres shorten after each cell division. Once a critically short length of telomeric DNA is reached, the cells enter cell arrest and eventually die. In contrast, telomerase activity is detectable in over 90% of known human tumor cells, enabling these tumor cells to escape senescence and to proliferate at a higher rate [3,4]. Thus, understanding telomerase biology and its complex regulation may shed light on how tumor cells acquire their capability for unlimited replication (immortality). Consequently, several therapeutic approaches to block telomerase activity have been suggested . The evaluation of all these novel therapies mainly depends on a reliable measurement of both the amount of telomerase protein and the enzymatic activity. Current technologies to study telomerase biology involve polymerase chain reaction (PCR) amplification for measuring telomerase activity [3,6] and Western blots for measuring telomerase proteins . Although highly sensitive, these techniques are time-consuming and can be prone to false-positive or false-negative results due to interferences and PCR artifacts . In addition, microarray methods for genomics and proteomics studies of telomerase are difficult to perform due to the multiple components required to measure enzyme activity.
Nanomaterials with unique magnetic and optical properties play an increasingly important role in the design of molecular probes for in vitro and in vivo diagnostics . In particular, nanoparticle-based assays that could quickly interrogate biologic systems and report on the amount of a particular enzyme, its level of activation, and its relation to disease state would have a tremendous impact in medicine. Recently, we have described the use of magnetic nanoprobes to sense for telomerase activity through changes in T2 water relaxation . We hypothesized that nanoparticle-based assays could be further refined to allow simultaneous detection of protein levels and enzymatic activities.
For our experiments, one set of magnetic nanoparticles was conjugated to synthetic oligonucleotides complementary to TTAGGG telomeric repeats, resulting in nanosensors able to measure telomerase activity (telomerase activity nanosensor). A second set was conjugated to a polyclonal anti-hTERT antibody, resulting in a nanosensor that detects telomerase protein (telomerase protein nanosensor; Figure 1). Using this dual nanosensor system, we were able to detect different amounts of telomerase protein and, concomitantly, measure telomerase activity in various cancer and normal cell lines. Most importantly, we were able to assess the contribution of phosphorylation on telomerase activity.
Aminated cross-linked iron oxide nanoparticles (CLIO-NH2 [10,11]) were conjugated to anti-hTER antibodies through protein G (Sigma-Aldrich, St Louis, MO). First, to conjugate protein G directly to magnetic nanoparticles, the aminated magnetic nanoparticles were precipitated in isopropanol and redissolved in DMSO to a concentration of 3.0 mg Fe/ml. Suberic acid bis(N-hydroxysuccinimide ester, DSS; Sigma-Aldrich) was reacted with 250 µl of the aminated magnetic nanoparticles (3.0 mg Fe/ml) for 1 hour at room temperature (125 mM DSS final concentration). The nanoparticles were washed three times by isopropanol precipitation, followed by dissolution in 100 µl of phosphate-buffered saline containing protein G (2 mg/ml). After an overnight incubation, the protein G-conjugated nanoparticles were purified using a magnetic separation column (Macs; Miltenyi Biotech, Auburn, CA) to separate unbound protein. The number of protein G molecules attached to the nanoparticle in solution was determined by a protein assay (Bradford assay). Finally, 500 µl of the antitelomerase antibody solution (H-231, 3 mg/ml; Santa Cruz Biotechnology, Santa Cruz, CA) was added to the protein G nanoparticle solution (500 µl, 1.5 mg Fe/ml) and incubated overnight at 4°C before purification by magnetic separation and washed with phosphate buffer (0.1 M pH 7.4). Typical final concentration of the magnetic nanoparticle solution was 0.5 to 0.9 mg Fe/ml.
Aminated cross-linked iron oxide nanoparticles (CLIO-NH2) were conjugated to a telomeric repeat specific oligonucleotide (Tufts University Core Facility, Somerville, MA) of sequence 5′-CCC-TAA-CCC-TAA-CCC-TAA-3′ [12,13] either with the 3′ or 5′ end, resulting in two particles (Telo-1 and Telo-2). Remaining free oligonucleotides were removed by magnetic purification of the Oligonucleotide-CLIO-NH2 (Miltenyi Biotec). Each CLIO particle had an average of four oligonucleotides bound. This number was determined by adding to Telo-1 and -2 a Cy-5-labeled complementary oligonucleotide (5′-TTA-GGG-TTA-GGG-3′) and by calculating the molar amount of fluorochrome bound to the particles using the extinction coefficient of Cy-5 at 648 nm.
Cell extracts were prepared from various cell lines (B16 melanoma, PC3 prostate cancer, hepatocellular carcinoma (HCC), 9L glioblastoma, E6-1 Jurkat lymphoma, OvCa ovarian carcinoma, and 5F rat insulinoma) by incubating approximately 1 x 106 cells in 200 µl of lysis buffer for 30 minutes on ice and then centrifuging at 12,000g for 30 minutes . Lysis buffer consisted of Tris-HCl (10 mM), MgCl2 (1 mM), EGTA (1 mM), CHAPS (0.5%), and PMSF (0.1 mM; all Sigma-Aldrich). Mouse tumor tissue samples of liver metastasis were homogenized in lysis buffer. Aliquots of the supernatant were stored at -80°C. The protein concentration (BCA; Pierce, Rockford, IL) varied between 0.5 and 4 mg/ml. For each telomerase assay, 1 µg of protein was used. Normal human primary tissue melanocytes were used as negative control and were a kind gift from Dr. Mark Eller, Department of Dermatology, Boston University School of Medicine [14–16]. To evaluate the state of telomerase activation by phosphorylation, cell extracts from selected cell lines were incubated at 37°C for 30 minutes in the presence or absence of 80 mU of protein phosphatase 2A (PPA; Calbiochem, San Diego, CA) as described previously . Telomerase activity was then determined as before.
T2 time relaxation measurements were carried out at 0.47 T and 40°C (Bruker NMR Minispec, Billerica, MA) in a total volume of 200 to 500 µl and with a final iron concentration of 10 µg Fe/ml with 1 µg of cell extract protein in 500 µl after the particles were incubated for 1 hour at 37°C. T2 values were obtained after adding varying amounts of synthetic telomeric repeats to determine the sensitivity of the assay. For magnetic resonance (MR) plate imaging, 50 µl of the assay was transferred into a 384-well plate with an iron concentration of 10 or 5 µg/ml (1:1 dilution). Plates were imaged in a 4.7-T small animal magnetic resonance imager (Pharmascan, Bruker) using T2-weighted spin-echo sequences with varying echo times to obtain a T2 map (repetition time (TR), 2000 milliseconds; echo time (TE), 25 to 200 milliseconds) and a T1-weighted (TR, 600 milliseconds; TE, 30 milliseconds) spin-echo sequence. Image T2 analysis was performed using OsiriX (www.osirix-viewer.com). A three-dimensional T2 image was constructed by fitting of a standard exponential transverse relaxation model (M0exp(-TE/T2)) to stacks of spin-echo MR image slices acquired at a TR of 2000 milliseconds and varying TE of 25 to 200 milliseconds. Renderings were performed at multiple angles to highlight the resolution and three-dimensional nature of the calculated T2 maps. Data are shown as δT2 (T2 of the blank minus T2 of sample) unless noted otherwise.
For Western blot analysis, tumor lysates were subjected to electrophoresis on SDS-PAGE followed by transfer to Hybond membrane (Amersham Biosciences, Piscataway, NJ) according to the manufacturer's instructions. Concentrations for blot analysis of anti-hTERT as well as secondary antibodies varied according to the manufacturers' recommendations (Santa Cruz Biotechnology; Jackson ImmunoResearch, West Grove, PA). The blots were developed with the ECL system (Amersham Biosciences). Immunoblot analysis of tubulin was used as a loading control (Sigma).
In the first set of experiments, we tested whether our anti-hTERT-nanosensors can detect telomerase protein accurately and reproducibly. The nanosensors were synthesized by conjugating an anti-hTERT antibody to dextran-coated iron oxide nanoparticles using protein G. Because human telomerase is difficult to purify from cell lysate  and purified telomerase is not commercially available, we first established by Western blot analysis that the antibody selected for conjugation to the nanoparticles indeed recognizes a protein in the size range of hTERT (120 kDa; Figure 2a) and compared the δT2 obtained with our nanosensors to the Western blot analysis of the telomerase present in various cell lines. Results show that the observed δT2 in the magnetic assays correlated well with the data obtained by Western blot (r2 = 0.9; Figure 2b). Next, we tested if our parallel telomerase activity nanosensor were able to interrogate telomerase activity (Figure 2c). Similar to the above described protein sensor and in agreement with previous results , validation experiments showed a tight fit between enzymatic activity measured by a commercial quantitative telomerase repeat amplification protocol (TRAP) and magnetic nanosensor measurements (r2 = 0.9; Figure 2c). Taken together, these results confirm that the nanosensor technology can indeed measure protein amounts and activity side by side in a simple and miniaturized format.
We subsequently determined whether the above measurements for telomerase protein can be made in parallel and at higher throughputs with either direct MR imaging (Figure 3) or benchtop relaxometry (Figure 4). We therefore analyzed eight different cell lysates from normal and tumor cell lines in quadruplicate, together with a number of internal controls. The measurement time for all samples was 20 seconds with MR imaging, and the detected signal intensities were very reproducible within the same cell line. No significant δT2 were observed for the negative controls, as expected. All tumor cell lines tested were positive for the presence of telomerase protein at various levels, whereas primary skin melanocytes tested negative (Figures 3 and and4).4). As a negative control, the corresponding cell lysates were preincubated with the anti-hTERT antibody to block the available hTERT epitopes before exposing them to the anti-hTERT nanosensors, observing no significant detection of telomerase enzyme (T2 signal) as expected. To exclude that the nanoparticles detected endogenous telomeric repeats already present in the extracts, we performed additional controls where cell extracts were either incubated with an unrelated primer or RNase experiments where the nucleotides were left out or experiments where the cell extracts were preheated to destroy telomerase activity . All these controls did not result in a significant change in the T2 time after incubating with the nanoparticles. Taken together, these results indicate that the observed changes in T2 are due to the interaction of the telomerase with the anti-hTERT antibody or the DNA on the particles and not due to nonspecific interaction with nontelomerase components in the cell lysate. Furthermore, the analysis can be performed rapidly with clinical MR scanners as shown in Figure 3.
We next compared levels of telomerase protein and telomerase activity within the same cell lines (Figure 4). Results show that HCC cells had the highest level of telomerase activity, whereas the melanoma cell line had a significantly lower telomerase activity. However, a higher amount of telomerase protein was present in the melanoma cell line compared to the HCC cell line. Also, a significantly lower amount of telomerase protein was present in insulinoma compared to lymphoma, whereas telomerase activity was much higher in the former. These observed discrepancies in protein levels and enzymatic activity are in line with previous reports that show that telomerase has different activation states. It has been shown that higher activity levels are necessary for sustained malignant growth and aggressiveness (ability to metastasize) [19–22]. These results indicate that telomerase can be present at different levels of activation and that measuring either telomerase activity or its activation alone might not be sufficient to obtain accurate information on the telomerase status of a given tumor.
We next investigated the apparent difference between the amount of hTERT protein and telomerase activity in the above mentioned cell lines. Specifically, we investigated the effects of phosphorylation by measuring the decrease in telomerase activity (indicated by δT2) upon incubation with PPA. The observed δT2 after PPA incubation should reflect the different native phosphorylation states of telomerase in these cell lines. As expected, different levels of activation of telomerase by phosphorylation were observed, ovarian and breast cancer cell lines having the most phosphatase-sensitive telomerase activity, which suggests that the telomerase in these cell lines is mostly activated by phosphorylation (Figure 5a). Interestingly, in the melanoma cell lines studied, phosphorylation also seems to play a role in the activation of the telomerase protein, although not as pronounced as in ovarian and cancer cells, which suggests different mechanisms in different cell lines.
The nanosensensor technology described here is based on magnetic nanoparticle conjugates, which form nanoclusters on target interaction. The nanocluster formation (typically 300–500 nm) causes a rapid decrease in the water T2 relaxation time . Because of the inherently built-in amplification (each cluster formation affects billions of surrounding water molecules), the ability to sense in turbid samples and the ability to design different assay configurations (DNA, RNA, protein, metabolites), this method provides unique advantages. Water relaxation can be readily detected by benchtop relaxometers  chemical nuclear magnetic resonance (NMR) systems, or even magnetic resonance imaging systems [17,24]. In this study, we evaluated whether parallel measurements could be made to determine protein levels and protein activity in biologic samples using the same readout, the proportional decrease in T2 (δT2). As a clinically relevant model system, we used telomerase, a protein implicated in tumorigenesis, whose functional state is affected differently by a number of drugs currently under development . We show that the developed technology can quickly and accurately determine the amount of telomerase protein and assess for its levels of activity and activation.
High levels of hTERT mRNA have been observed in most tumors with high telomerase activity , suggesting that hTERT is by itself an up-regulating factor in its own activation, essential in oncogenesis, tumor progression, and tumor invasion . However, some lesions and normal tissue with low or undetectable telomerase activity have been found to contain significant levels of hTERT mRNA, presumably due to either alternate splicing  or posttranslational modifications such as phosphorylation-dephosphorylation [17,29–31] that might alter the activity of the enzyme. Most importantly, high levels of telomerase activity have been reported in aggressive tumors and in metastatic lesions presumably due to up-regulation of telomerase reverse transcriptase . However, the effect of phosphorylation on the telomerase activity of these metastatic lesions is not well understood at this time.
On the basis of our results, the measured activity of telomerase has two main components: 1) the contribution from the total amount of telomerase protein and 2) the level of phosphorylation of telomerase protein. When both of these parameters are taken into account by multiplying the detected amount of telomerase protein (as the δT2 protein) with the amount of phosphorylation-induced activation of telomerase (δT2 activation) and plotted against the measured telomerase activity (δT2 activity), a linear correlation was observed (Figure 5b). This observation has important implications for the evaluation of novel antitelomerase therapies because both the level of activation and the amount of hTERT protein must be taken into account. It is thus highly advisable to determine not only the activity but also the amount of protein to determine whether a given telomerase activity level is either due to highly activated telomerase present in small amounts or to large amounts of less active enzyme, because this could have important implications for an antitelomerasegeared therapy.
In summary, we have developed an integrated nanosensor technology to determine the levels of a neoplastic protein marker (telomerase) and its level of activation in various cancer cells lines. With this method, results can be obtained more quickly, thereby using the same nanoparticle technology platform, instead of laborious and costly methods such as reverse transcription-PCR and Western blots. The method can be easily adapted to study other cancer-related protein markers such as metalloproteinases and caspases, among others. Current developments in NMR miniaturization  and portable relaxometers  will facilitate the implementation of the described technique in future cancer diagnostics, prognostics, and the assessment of antitelomerase-geared therapies.
The authors thank the help of Nikolay Sergeyev for bulk synthesis of magnetic nanoparticles and Claire Kaufman for technical assistance with MR imaging.
1This work was supported in part by the National Institutes of Health grants R24-CA92782, R21/R33 CA91807, U54-CA119349, P50-CA86355, U54 CA126515 (R.W.), and KO1 (CA101781) to J.M.P. as well as R25CA096945-05 (J.G.).