Thyroglobulin may affect telomerase activity in thyroid follicular cells

Figures & data

Figure 1.  (a) Telomerase activities in HeLa cell extract. Each value is the mean of duplicate experiments. ○, positive control in the assay kit; •, HeLa cell extract. (b) Effect of human thyroglobulin on telomerase activity in vitro. Each value is the mean of duplicate experiments. 10⁶ HeLa cells extract/assay tube were used as telomerase enzyme. ○, 0% 66,000 MW bovine serum albumin (BSA) instead of thyroglobulin was added as a control; ⊚, 0.1% (final) BSA; ▪, 1% (final) BSA.

Table I.  Effect of iodine or thyroid hormones and or human thyroid extract on telomerase activity.

Telomerase in the assay	Final concentration of T3, T4, iodine and albumin and or solvents or thyroid extract	T3		T4		Iodine	Thyroid extract^*	Albumin	Distilled water
		in DMSO	in NaOH	in DMSO	in NaOH
+	10^− 3 M	2.25 (A450 nm)	2.30	2.87	3.32
+	10^− 4 M	2.75	3.30	2.53	2.99
+	10^− 5 M	3.18	2.92	2.43	3.21
+	10^− 6 M	3.11	3.10	2.36	3.30
+	10^− 7 M	3.01	–	2.82	–
+	0.5% DMSO only	3.20	–	3.20	–
+	0.001 N NaOH only	–	3.33	–	3.33
+	10^− 3 M					3.01
+	10^− 5 M					2.54
+	10^− 7 M					3.24
+	2% extract						0.07
+	5% extract						0.06
+	0.1% (final)							3.01
+	1% (final)							2.88
+ (posit.)	0								3.35
− (neg.)	0								0.02

Each value is the mean of duplicate or triplicate experiments. DMSO: dimethylsulfoxide, T₃: B-triiodothyronine, T₄: B-thyroxine. T₃ and T₄ were dissolved in 0.5% DMSO or 0.001 N NaOH. Telomerase was extracted from 10⁶ Hela cells harvested. Posit, or neg: positive or negative control prepared in the assay kit. A: absorbance. *Three thyroid tissues of Graves' disease or 2 thyroid tissues adjacent to thyroid adenoma or 2 normal thyroid tissues were homogenized with 20% or 50% (W/V) lysis buffer (phosphate buffer and saline (pH 7.5), or saline) and the homogenates were centrifuged at l000 × g for 10 min. The supernatants were used for the assay. The values are the mean of duplicate or triplicate experiments.

Figure 2.  Effect of intermediate addition of dNTP(a) or TS primer(b) on the telomerase assay. Each value is the mean of duplicate experiments. HTg, human thyrogrobulin; posit., positive control; neg., negative control.

Figure 3.  Effect of human thyroglobulin on ordinary or real time quantitative PCR product (particularly taq DNA polymerase). (a) ordinary PCR. Lane 1 ∼ 6, UCSNP 19 is used as a primer ( ← ); M, size marker; 1: control: without addition of human thyroglobulin. 2 ∼ 6: addition of 10^{− 9}∼10^{− 5} (ten-fold serially diluted) M of human thyroblobulin at the beginning of PCR. (b) real time quantitative PCR. The primer used was TaqMan Gene Expression Assay (ID: Hs00355773-ml). At the beginning of the real time PCR 10^{− 5} M (red line) or 10^{− 6} M (green line) and or 10^{− 7} M (blue line) of human thyroglobulin. Purple line: without addition of human thyroglobulin at the beginning of the PCR (positive control). Dotted red line under the threshold line (dark green): without addition of template DNA at the beginning of the PCR (negative control). Both positive and negative control experiments were successfully proceeded.