Supplementalmaterials and Methods

SupplementalMaterials and methods

Materials

Wherever possible, nuclease-free buffers and reagents were used to minimize RNase contamination. RNase-free water, 2.0 M potassium acetate (AcOH-AcOK, pH 5.5), 3.0 M KCl and 5.0 M NaCl, oligo(dT)12-18, dNTP mix, 5× first-strand buffer (250 mM Tris-HCl, pH 8.3, 375 mMKCl, 15 mM MgCl2), SuperScript reverse transcriptase II, 1x FAST SYBR Green Master Mix, MEM 11095, and RPMI 1640 were purchased from Life Technologies (Carlsbad, CA). Concentrated phosphate-buffered saline or 10 × PBS (81 mM Na2HPO4, 14.7 mM KH2PO4, 1.37 M NaCl, 26.8 mMKCl) was obtained from Nippon Gene (Tokyo, Japan). The lysis buffer comprised of 10 mM Tris-HCl (pH 7.5), 1.0 mM MgCl2, 1.0 mM benzamidine, 5.0 mM 2-mercaptoethanol (2-ME), 0.5% 3-[(3-cholamidopropyl)-dimethylamino]-1-propanesulfonate (CHAPS) and 10% glycerol was purchased from EMD Millipore (Billeria, MA). Taq polymerase was purchased from Promega (Madison, WI). Recombinant RNase Inhibitor from porcine liver (Takara Bio, Shiga, Japan) was added to RNase-free water to 20 units/μL. GelStar® Nucleic Acid Stain was obtained from Takara Bio. FND was the same as that used previously (1). The ETS-Primer containing a disulfide group for immobilization on the electrode, HO-(CH2)6-SS-(CH2)6-5’ TTT TTT TTA ATC CGT CGA GCA GAG TTA GGG-3’, and RT-PCR primers were custom synthesized by Genenet (Fukuoka, Japan). The sequences of the latter were as follows: glyceraldehyde phosphate dehydrogenase (GAPDH) forward, 5’-ATGGAAATCCCATCACCATCTT-3’ (Tm, 54.0 °C, estimated by Primer Express Software Version 3.0, Life Technologies) and reverse, 5’-CGCCCCACTTGATTTTGG-3’ (Tm, 54.5 °C); human telomerase reverse transcriptase (hTERT) forward, 5’-CTGCTGCAGAAATAGCGTCATT-3’ (Tm, 58.0 °C) and reverse, 5’-AAAGCGAGAGCAGCAAAGCT-3’ (Tm, 59.0 °C). HeLa cells and the TRAPese kit were obtained from EMD Millipore. HSC-2 cells, human oral squamous cell carcinoma cell lines, and HSC-3 cells, human tongue squamous cell carcinoma cell lines, Ca9-22 cells, human gingival squamous cell carcinoma cell lines, and SAS cells, and human tongue squamous cell carcinoma cells were purchased from Riken Bioresource Center (Ibaraki, Japan). Proteostain Protein Quantification Kit-Wide Range was obtained from Dojindo (Kumamoto, Japan). RNeasy kit was purchased from Qiagen (GmbH, Hilden, Germany).

Apparatus

Ultraviolet-visible spectra were obtained on an ND-1000 (Thermo Fisher Scientific, Waltham, MA). Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) was performed with the StepOne™ Real-Time PCR System (Applied Biosystems). Osteryoung square wave voltammetry (SWV) were measured on an ALS 650C (CH Instrument, Austin, TX) with an adapter for the disposable electrode. Before SWV measurement the electrode was incubated at -0.16 V for 1 s, at -0.55 V for 1 s and at -0.16 V for 1 s by the multi potential step mode. SWV was also measured with an initial voltage of 0 V, final voltage of 0.5 V, applied potential of 10 mV, amplitude of 50 mV, frequency of 70 Hz, and sensitivity 1 x 10-5 A/V.

Quantitative real-time RT-PCR

Total RNA was prepared from cells with an RNeasy kit, according to the manufacturer’s protocol. Purified RNA was eluted in 30 μL of RNase-free water and stored at -80 °C until use. The amount of RNA was measured spectrophotometrically on an ND-1000 based on the molar extinction coefficient of 40 ng-cm/μL at 260 nm. To 1 μg of RNA (1.8, 1.3, 1.6, 1.7 and 0.7 μL for HeLa, HSC-2, HSC-3, SAS and Ca9-22, respectively) was mixed with 1.0 μL of 500 μg/mL oligo(dT)12-18 and 1.0 μL of 10 mMdNTP mix, and diluted to 13 μL with RNase-free water. The mixtures were heated to 65 °C for 5 min and chilled quickly. After addition of 4.0 μL of 5× first-strand buffer and 2.0 μL of 0.1 M dithiothreitol, they were incubated at 42 °C for 2 min. SuperScript reverse transcriptase II was then added to synthesize cDNA at 42 °C for 50 min, followed by enzyme inactivation at 70 °C for 15 min. cDNA thus prepared was stored at -20 °C until use.

The reaction solution contained 2.0 μl of cDNA product, 0.2 μM each PCR primers (hTERT forward and reverse primers, glyceraldehyde 3-phosphate dehydrogenase, GAPDH, as a house keeping gene, forward and reverse primers), 1x FAST SYBR Green Master Mix in 20 μL. Thermal cycling was carried out on a StepOne™ Real-Time PCR System under the following conditions: 95 °C for 20 s; 40 cycles of 95 °C for 30 s; and 60 °C for 30 s, with fluorescence quantification at 60 °C. The data obtained were analyzed by software StepOne linked directly with the instrument. The Ct value used for quantification of hTERT expression represents the PCR cycle number that gives the lowest fluorescence intensity detectable and reflects the initial amount of cDNA. Specifically, the ΔRn values, which are an increment of fluorescence signal at each time point, were plotted against the cycle number and the Ct is where ΔRn is 0.83. The fold increase in copy numbers of mRNA was determined relative to GAPDH.

TRAP assay

TRAP assay was performed with a TRAPeze kit (EMD Millipore) according to the manufacturer’s instructions. The telomerase reaction solution (25 μL) consisted of 20 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 63 mMKCl, 0.05% Tween 20, 1.0 mMethyleneglycol-bis(ß-aminoethyl)-N,N,N’,N’-tetraacetic acid, 50 μM each dNTP mixture, 1× TS primer, 1× primer mix, 2.0 units of Taq polymerase, 2.0 μg of the lysate protein.Reactions were run under the following conditions: 1× 30 °C, 1 h; 33× (95 °C, 30 s, 59 °C, 1 min, 72 °C, 30 s); 1× 72 °C, 20 min. Gel electrophoresis on 12.5% polyacrylamide prepared in 1.25× TBE (89 mM Tris base, 89 mM borate, and 1 mM ethylenediaminetetraacetic acid, pH 8.0) was run at 200 V for 1 h in 0.7× TBE. After electrophoresis, the gel was stained with 1× GelStar® Nucleic Acid Stain in 1× TBE for 30 min and photographed. Where no elongation was observed beyond 50 bp of the TS primer (internal standard), samples were judged negative (-). Where the number of addition of the TTAGGG sequence was 1 - 4, they were judged ambiguous (±), and where the number was greater than 4, they were judged positive (+).

References

Sato S, Takenaka S. Linker effect of ferrocenylnaphthalene diimide ligands in the interaction with double stranded DNA , J OrganometChem 2008; 693: 1177-85.

Supplemental Data Fig. 1.Telomerase activities of cell line SAS analyzed by TRAP using 0- (a), 10- (b), 50- (c), 75- (d), 100- (e) and 200-cell extracts (f). M stands for DNA size markers and internal standard and the arrow for the internal standard (36 bp) and the primer of 50 bp, respectively.

Supplemental Data Table 1.Telomerase activity of 20 patients with oral squamous cell carcinoma.

No. / Age / Sex / Position / Tissue size
W×H×D / cm / Exfoliated oral cells / SCC tissue
[protein]/
μg/μL / Δi/% / TRAP / [protein] /
μg/μL / Δi/% / TRAP
1 / 68 / F / tongue / 1.5×1.0×0.8 / 0.25 / 58 / - / 2.0 / 41 / ±
2 / 76 / M / gingiva / 5.0×2.8×2.5 / 7.0 / 18 / - / 4.2 / 62 / ±
3 / 86 / M / gingiva / 3.0×1.2×2.0 / 0.29 / 30 / ± / 1.8 / 39 / ±
4 / 71 / F / gingiva / 2.5×1.0×0.8 / 0.19 / 23 / - / 1.0 / 24 / ±
5 / 76 / F / gingiva / 1.7×1.5×2.2 / 1.3 / 40 / - / 0.53 / 66 / -
6 / 34 / F / tongue / 1.1×0.8×0.6 / 0.32 / 100 / - / 1.3 / 33 / +
7 / 69 / M / gingiva / 3.0×2.0×2.0 / 0.61 / 46 / ± / 1.9 / 37 / +
8 / 76 / F / gingiva / 1.3×1.3×1.0 / 1.3 / 39 / - / 1.6 / 37 / +
9 / 61 / M / tongue / 1.2×1.6×0.6 / 1.3 / 48 / ± / 3.1 / 42 / ±
10 / 53 / M / tongue / 1.5×1.0×1.5 / 1.2 / 44 / - / 4.1 / 61 / +
11 / 67 / M / tongue / 2.0×1.5×4.0 / 0.43 / 56 / - / 1.7 / 51 / +
12 / 59 / M / tongue / 6.0×6.0×5.5 / 3.4 / 51 / - / 3.3 / 18 / -
13 / 68 / M / tongue / 4.2×2.8×2.4 / 2.4 / 52 / - / 3.3 / 59 / +
14 / 43 / M / tongue / 1.5×1.0×1.0 / 0.32 / 39 / - / 2.1 / 45 / ±
15 / 68 / M / tongue / 3.2×2.1×1.8 / 0.39 / 28 / - / 0.49 / 34 / ±
16 / 82 / M / gingiva / 1.2×1.0×1.0 / 1.4 / 53 / + / 3.9 / 50 / ±
17 / 70 / F / tongue / 2.0×1.0×2.0 / 2.3 / 64 / - / 1.9 / 37 / +
18 / 60 / M / gingiva / 1.3×0.6×1.3 / 3.3 / 44 / ± / 3.7 / 60 / +
19 / 75 / F / gingiva / 0.9×0.2×0.5 / 3.6 / 32 / + / 4.2 / 63 / +
20 / 78 / F / buccal mucosa / 0.8×1.0×0.1 / 1.1 / 30 / - / 1.6 / 30 / +

Supplemental Data Table 2. Telomerase activity of 27healthy volunteers.

Case / Age / Sex / Exfoliated oral cells / Tissue
(buccal mucosa) / Tissue (tongue)
[protein] /
μg/μL / Δi/% / TRAP / [protein]/
μg/μL / Δi/% / TRAP / [protein] /
μg/μL / Δi/% / TRAP
21 / 24 / F / 3.1 / 1.9 / - / 2.1 / 9.8 / - / 2.7 / 13 / -
22 / 28 / F / 1.8 / 12 / + / 3.1 / 13 / - / 2.8 / 3.0 / -
23 / 29 / M / 1.8 / 11 / - / 2.4 / 33 / - / 2.7 / 15 / -
24 / 25 / F / 1.8 / 13 / - / 2.9 / 13 / - / 3.3 / 15 / -
25 / 28 / M / 1.1 / 19 / na / 1.5 / 17 / - / 0.93 / 5.9 / -
26 / 27 / M / 0.15 / 26 / na / 0.90 / 3.4 / - / 1.1 / 3.9 / -
27 / 27 / M / 0.67 / 18 / na / 1.4 / 6.4 / - / 1.1 / 13 / -
28 / 32 / M / 1.1 / 37 / na / 1.2 / 1.9 / ± / 1.4 / 12 / -
29 / 27 / M / 0.86 / 12 / - / 2.5 / 16 / - / 0.28 / 14 / ±
30 / 30 / M / 4.5 / 12 / na / 1.3 / 6.8 / - / 2.4 / 15 / -
31 / 33 / M / 0.40 / 25 / -
32 / 41 / M / 2.9 / 18 / -
33 / 53 / M / 4.1 / 17 / -
34 / 35 / M / 0.22 / 18 / -
35 / 53 / M / 0.62 / 8.4 / -
36 / 35 / F / 0.66 / 11 / -
37 / 80 / F / 4.0 / 23 / -
38 / 65 / F / 4.4 / 16 / -
39 / 77 / M / 6.6 / 18 / ±
40 / 71 / F / 6.9 / 10 / -
41 / 45 / M / 4.7 / 14 / -
42 / 75 / M / 4.5 / 17 / ±
43 / 76 / F / 4.0 / 4.9 / ±
44 / 60 / F / 5.3 / 16 / ±
45 / 73 / M / 5.2 / 14 / ±
46 / 84 / F / 5.8 / 13 / ±
47 / 90 / F / 4.4 / 127 / +

Supplemental data Table 3.Telomerase activity of SAS, Ca9-22, HSC-2 and HSC-3 cells as revealed by TRAP. Where the number of addition of TTAGGG was 1 - 4, samples were judged ambiguous (±), and where it was greater than 4, they were judged positive (+). Where no ladders were observed, they were judged negative (-).

Number of cells / SAS / Ca9-22 / HSC-2 / HSC-3
0 / - / - / - / -
10 / - / - / - / -
50 / ＋ / - / - / -
75 / ＋ / ± / ± / ±
100 / ＋ / ± / ＋ / ＋
200 / ＋ / ＋ / ＋ / ＋

Supplemental data Table 4.Specific telomerase activity determined by ECTA.

[protein]/ ng/µL / Δi/% / Δi /Δi- HeLa / Δi/[protein] / mRNA for hTERT
SAS / 130 / 21.4 / 0.95 / 1.64 / 6.33
Ca9-22 / 100 / 7.4 / 0.33 / 0.74 / 0.87
HSC-2 / 100 / 16.6 / 0.74 / 1.66 / 2.37
HSC-3 / 80 / 18.9 / 0.84 / 2.36 / 3.49
HeLa / 200 / 22.4 / 1.00 / 1.00

Supplemental data Table5. Positive rate for oral cancer with tumor size

Tumor
Size (cm) / Number of “+” and “±” / Tissue / Exfoliated oral cells
TRAP / ECTA / TRAP / ECTA
<2.0 / 11 / 11 (100%) / 11 (100%) / 4 (36%) / 11 (100%)
≥2.0 / 9 / 7 (78%) / 8 (89%) / 2 (22%) / 8 (89%)

Numbers in the parentheses represent the positive rate defined by the number of “+” and “±” divided by the patient number.

Supplemental data Table 6.Blind test by ECTA.

No. / Δi / % / C/Ha / Hit / Comments / No. / Δi/% / C/Ha / Hit / Comments
1 / 78 / C / yes / 29 / 18 / H / yes
2 / 32 / H / no / f.p. / 30 / 22 / H / yes / ±
3 / 41 / C / yes / 31 / 27 / H / yes / ±
4 / 64 / C / yes / 32 / 32 / H / no / f.p.
5 / 31 / C / yes / 33 / 99 / C / yes
6 / 28 / H / yes / ± / 34 / 44 / H / no / f.p.
7 / 25 / C / no / f.n. / 35 / 47 / C / yes
8 / 22 / C / no / f.n. / 36 / 2 / H / yes
9 / 9 / H / yes / 37 / 28 / H / yes / ±
10 / 12 / H / yes / 38 / 62 / C / yes
11 / 29 / H / yes / ± / 39 / 14 / H / yes
12 / 38 / C / yes / 40 / 35 / C / yes
13 / 27 / H / yes / ± / 41 / 2 / H / yes
14 / 23 / H / yes / ± / 42 / 23 / H / yes / ±
15 / 11 / H / yes / 43 / 37 / C / yes
16 / 33 / C / yes / 44 / 20 / H / yes / ±
17 / 27 / H / yes / ± / 45 / 6 / H / yes
18 / 51 / C / yes / 46 / 32 / C / yes
19 / 30 / C / yes / 47 / 15 / C / no / f.n.
20 / 1 / H / yes / 48 / 10 / H / yes
21 / 44 / C / yes / 49 / 29 / C / no / f.n.
22 / 45 / C / yes / 50 / 46 / C / yes
23 / 48 / C / yes / 51 / 34 / C / yes
24 / 56 / C / yes / 52 / 31 / H / no / f.p.
25 / 17 / C / no / f.n. / 53 / 48 / C / yes
26 / 51 / C / yes / 54 / 41 / C / yes
27 / 39 / C / yes / 55 / 44 / C / yes
28 / 12 / H / yes / 56 / 60 / C / yes

aC means cancer patients, H means Healthy volunteers.