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The activity of thymidine kinase (TK – EC 2.7.1.21) was measured in human thyroid tissue homogenates incubated in vitro. This enzyme functions as a part of the ...
ENDOCRINE REGULATIONS, Vol. 32, 9 – 16, 1998

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THYMIDINE KINASE (EC 2.7.1.21) ACTIVITY IN HOMOGENATES OF HUMAN THYROID TISSUE, FOLLOWING THE EXPOSURE TO EPIDERMAL GROWTH FACTOR (EGF) IN VITRO J. BRZEZINSKI1, M. KARBOWNIK2, A. GESING2, A. LEWINSKI2, M. KLENCKI2, H. MODRZEJEWSKA3, J. GREGER3 Department of Endocrine Surgery, 2Department of Thyroidology, Institute of Endocrinology and 31st Department of Biochemistry, Medical University of Lodz , Lodz , Poland

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The activity of thymidine kinase (TK – EC 2.7.1.21) was measured in human thyroid tissue homogenates incubated in vitro. This enzyme functions as a part of the pyrimidine salvage pathway involved in DNA synthesis. The thyroid tissue was obtained from the thyroids of patients thyroidectomized at the Department of Endocrine Surgery, Medical University of Lodz because of: 1. non-toxic nodular goiter (NTNG): tissue macroscopically unchanged, woman 46 yrs; 2. nontoxic adenoma (NTA), woman 37 yrs; 3. toxic adenoma (TA), woman 45 yrs. The tissue was incubated for 4 hours in RPMI 1640 medium (Gibco), containing Hepes buffer, 15 % FCS and the examined substance – epidermal growth factor (EGF) – used in five different concentrations (0.1 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, 1000 ng/ml). The TK activity was measured according to CHENG and PRUSOFF (1974) as modified by GREGER and DRAMINSKI (1989). The reaction products were separated by ascending chromatography. It was found that: 1. TK activity in thyroid tissue from NTNG and NTA did not significantly differ from control incubations without EGF, while it was significantly higher in the thyroid tissue from TA; 2. in the range of concentrations from 1 ng/ml to 1000 ng/ml, EGF increased TK activity in the macroscopically unchanged tissue from NTNG; 3. the incubation of the tissue from NTA with EGF resulted in the increase of TK activity in a concentration-dependent manner; 4. EGF stimulated TK activity in the tissue from TA, but the difference was significant only after the lowest EGF concentration. The obtained results suggest a possible role of EGF in the pathogenesis of NTNG, as well as of NTA and TA in humans. Key words: Human thyroid – Thymidine kinase (EC 2.7.1.21) – EGF – Incubation in vitro

Epidermal growth factor (EGF) is a mitogen for various cells and tissues (GOSPODAROWICZ et al. 1978), among others, for thyroid follicular cells (TFC). That growth factor was demonstrated to stimulate the TFC proliferation in cell cultures prepared from thyrocytes of various animal species (ROGER and DUMONT 1982; WESTERMARK and WESTERMARK 1982; WESTERMARK et al. 1983) and also in suspension culture of porcine thyroid follicles (GÄRTNER et al. 1985). On the other hand, some authors failed to observe the proliferogenic effect of EGF on rat thyrocytes (SMITH et al. 1986), first of all, on the cultured FRTL-5 cells (AMBESI-IMPIOMBATO et al. 1980; VENEZIANI et al. 1986; PANG

and HERSHMAN 1990). In contrast, however, some authors observed even the stimulation of FRTL-5 cell line proliferation after EGF (ASMIS et al. 1995). Also in our laboratory, EGF was previously shown to stimulate TFC mitogenesis in the organ culture of rat thyroid lobes (ZEREK-MELEN et al. 1990). On the other hand, in our recent experiments, EGF, injected in vivo directly into rat thyroid lobes (BRZEZINSKI et al. 1996), or added into an incubation medium (BRZEZINSKI et al. 1995), did not significantly affect the rate of 3Hthymidine incorporation into thyroid DNA. However, the stimulatory effect of EGF on TFC growth processes was confirmed in the experiments in vivo, after

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EGF AND THYMIDINE KINASE IN HUMAN THYROID

xenotransplantations of rat (OZAWA and SPAULDING 1990) or human thyroid tissue (HOELTING et al. 1994; PASCHKE et al. 1995) into nude mice. Deoxythymidine kinase [thymidine kinase (TK) – ATP : thymidine 5'-phosphotransferase, EC 2.7.1.21) is the enzyme responsible for catalyzing the phosphorylation of thymidine, which functions as a part of the pyrimidine salvage pathway, involved in DNA synthesis (KAHN et al. 1980). The activity of TK is closely correlated with 3H-thymidine incorporation into DNA of various tissues and with cell divisions (KAHN et al. 1980, ZIEVE et al. 1985). The increased TK activity was found in thyrocytes of autonomous nodules, when compared with normal thyroid tissue (MUELLER-GAERTNER et al. 1989). Moreover, the increased TK activity was demonstrated in the thyroids of patients with Graves’ disease, nontoxic nodular goiter (NTNG), adenomas and, especially, with thyroid cancers (MURAKAMI 1988). In another experiment, TK activity was measured in homogenates of rat thyroid lobes, incubated in vitro with EGF and, unexpectedly, we found that EGF – in all the applied concentrations – decreased TK activity (BRZEZINSKI et al. 1997). So far, certain discrepancies in EGF effects on the growth processes in the thyroid of several species as well as in the human thyroid have not been sufficiently explained. In this study, we have attempted to examine the effects of EGF on TK activity in homogenates of human thyroid tissue in vitro, collected – during the surgery – from patients with NTNG (fragments macroscopically unchanged), as well as with non-toxic adenoma (NTA) or toxic adenoma (TA). Materials and Methods The thyroid tissue was obtained from patients subjected to subtotal thyroidectomy at the Department of Endocrine Surgery, Institute of Endocrinology, Medical University of  Lodz. There were three different types of the thyroid tissue, diagnosed as: 1. non-toxic nodular goiter (NTNG), tissue macroscopically unchanged, obtained from a woman (46 yrs); postoperative histopathological diagnosis: “nodular goiter”; 2. non-toxic adenoma (NTA) obtained from a woman (37 yrs), postoperative histopathological diagnosis: “follicular adenoma”;

3. toxic adenoma (TA) – obtained from woman (45 yrs) who was treated before the surgery by antithyroid drugs because of thyrotoxicosis and recovered to the euthyroid state prior to subtotal thyroidectomy; postoperative histopathological diagnosis: “follicular adenoma”. The thyroid tissue was weighed on a torsion balance and, immediately, placed into incubation fluid. The tissue was incubated for 4 hrs in RPMI 1640 medium (Gibco), containing Hepes buffer, 15 % fetal calf serum (FCS), penicillin (200 U/ml), streptomycin (10 µg/ml) and EGF (Sigma, St. Louis, MO), used in five different concentrations (0.1 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, 1000 ng/ml). The tissue mass in each group (i.e. each of three types of thyroid tissue from the patients, controls plus five concentrations of EGF in each) was approximately the same (the mean and S.D. of all groups was 308.2 ± 7.9 mg, range 300.0 – 328.0 mg). The control incubations were conducted without any addition of EGF to the medium. Subsequent steps of the procedure were carried out according to CHENG and PRUSOFF (1974) as modified by GREGER and DRAMINSKI (1989). After the incubation, thyroid lobes of the particular groups were homogenized in the medium (25 mM Tris-HCl buffer, 25 mM KCl and 5 mM MgCl2 – pH 7.4, temp. 0 o C). Following centrifugation (10000 x g for 20 min), the obtained postmitochondrial fraction (70 µl) was incubated for 45 min (37 oC) in the medium consisting of: 50 mM Tris-HCl buffer (pH 7.4), 10 mM ATP, 10 MgCl2 and, additionally, with 35 µl [2-14C]dThd. The reaction was terminated by immersing in a boiling water bath (100 oC, 2 min). After deproteinization (by centrifugation – 3 min), the aliquots of supernatant were placed on Whatman No. 1 chromatographic paper. The reaction products – dTMP and dThd were separated by ascending chromatography at room temperature in a solvent of n-butanol : acetone : 5 % ammonia : glacial acetic acid : H2O (90 : 30 : 20 : 20 : 40, V/V). Five (5) parallel chromatographic separations for each group were conducted. The chromatograms were dried and the radioactive spots, corresponding to dTMP and dThd, were cut out and placed in counting vials. Radioactivity was measured in an LKB Wallac liquid scintillation counter. The protein content was determined by means of the biuret method (GORNALL et

EGF AND THYMIDINE KINASE IN HUMAN THYROID

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Fig. 1 Thymidine kinase activity in homogenates of the macroscopically unchanged thyroid tissue obtained from the patient with non-toxic nodular goiter (NTNG) and incubated in vitro in the presence of EGF. Bars represent means ± S.E.M., P – level of significance; a – P