Hormonal regulation of thyroid peroxidase in normal and transformed rat thyroid cells.

Hormonal Regulation of Thyroid Peroxidase In Normal And Transformed Rat Thyroid Cells

Raffaele Zarrilli, Silvestro Formisano, and Bruno Di Jeso Centro di Endocrinologia ed Oncologia Sperimentale del C.N.R., Dipartimento di Biologia e Patologia Cellulare e Molecolare (R. Z., S. F., B. Di. J.) II Facolta di Medicina e Chirurgia Napoli, Italy Istituto di Biologia (S.F.) Facolta di Medicina Udine, Italy

4), TPO expression (4-8) and I" uptake (4, 9, 10). In the continuous rat thyroid cell line FRTL-5 (11), TSH is not the only stimulator of thyroid function: insulin and insulin-like growth factor I (IGF-I) also positively regulate TG synthesis (12). The question arises as to whether insulin and IGF-I also regulate TPO expression and I" uptake in FRTL-5 cells It is known that several oncogenes act negatively on the expression of TG and on the I" uptake (13,14). In an in vitro model of tumor progression developed in a rat thyroid differentiated cell line, the PC cl.3 cells, cmyc, or polyomavirus middle-T-antigen (PyMLV) repress I" uptake, leaving unaltered TG synthesis; cooperation between the two oncogenes, however, suppresses both thyroid differentiated functions (13). Suppression of TG synthesis and I" uptake is also observed when thyroid cells are transformed by ki-ras oncogene (FRTL-5 KiMol) (14). However, it is not known whether neoplastic transformation affects TPO expression. In this study, we have investigated the effect of insulin and IGF-I on TPO mRNA levels and on I" uptake in FRTL-5 cells. We demonstrate that insulin and IGF-I, as well as TSH, positively regulate TPO mRNA levels, while they are completely ineffective on I" uptake. We have also analyzed the expression of TPO after c-myc or PyMLV transformation of PC cl.3 cells. We show that 1) cooperation between c-myc and PyMLV represses l~ uptake as well as TPO and TG mRNAs; 2) PyMLV alone represses TPO mRNA and I" uptake leaving unaltered TG mRNA; 3) c-myc decreases only I" uptake.

The hormonal induction of thyroid peroxidase (TPO) mRNA is studied in the functional rat thyroid cell line FRTL-5 and compared to the induction of thyroglobulin (TG) mRNA and I" uptake . TPO and TG mRNAs are regulated by TSH and by insulin-like growth factor I (IGF-I) and/or insulin. However, while TPO is more sensitive to TSH regulation (5- to 6-fold increase vs. 2- to 3-fold increase by IGF-I), TSH and IGF-I are equally potent in increasing TG mRNA levels (3- to 4-fold). Regulation of I" uptake appears to be different: thus TSH greatly (15-fold) increases I" uptake, while IGF-I or insulin are completely ineffective. TPO and TG mRNAs and l~ transport display different sensitivity to transformation of rat thyroid cells. Thus, when another differentiated rat thyroid cell line, the PC cells, are transformed by human cmyc (PC myc), TPO and TG mRNAs are both present at normal levels, while I" uptake is slightly decreased; in the PC cells transformed by polyomavirus middle-T-antigen (PC PyMLV) TPO mRNA is undetectable and I" uptake is greatly decreased, while TG mRNA is present at normal levels. All three differentiated functions are switched off in PC cells transformed by the cooperation of c-myc and polyomavirus middle-T-antigen (PC myc + PyMLV). (Molecular Endocrinology 4: 39-45, 1990)

INTRODUCTION

The function of the thyroid gland, necessitates the expression of specific molecules, i.e. thyroglobulin (TG), thyroid peroxidase (TPO), I" carrier, that are required for the synthesis of thyroid hormones (1). TSH, an important regulator of thyroid function, has been shown to stimulate, both in vivo and in vitro, TG synthesis (2-

A Rat TPO cDNA Probe Identifies Tissue Specific mRNAs in the Rat Thyroid

0888-8809/90/0039-0045$02.00/0 Molecular Endocrinology Copyright © 1990 by The Endocrine Society

When total RNA is hybridized to a 0.7 kilobase (kb) EcoRI-SacI probe corresponding to the 5'-coding re-

RESULTS

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MOL ENDO-1990 40

gion of the rat TPO cDNA signals appear between the 28 S and 18 S markers, both in FRTL-5 cells and in thyroid tissue (FIG. 1A). In lane 3 of Fig. 1A there are two additional bands migrating above the 28 S marker which reflect, most likely, incomplete denaturation of the abundant message. No hybridization is detected using comparable amounts of RNA prepared from rat heart (lane 5), liver (lane 6), kidney (lane 7), brain (lane 8), testis (lane 9), and spleen (lane 10). Densitometric analysis of the above Northern blot autoradiographs shows that TPO mRNA levels are 10-fold higher in rat thyroid tissue than in FRTL-5 cells. As shown in Fig. 1B, two mRNA transcripts of about the same size reported by others (6,8,15) are detected using poly(A)+ RNA preparations of FRTL-5 cells (lane 1, 10 ^g) and of rat thyroid (lane 2, 2 ^g)- The relative ratio of the two mRNA species is different in rat thyroid tissue compared to FRTL-5 cells. IGF-I and/or Insulin, as well as TSH, Increase TPO mRNA Levels in FRTL-5 Cells When FRTL-5 cells are depleted of TSH, insulin and serum supplement for 5 days (see Materials and Methods), TPO expression is inhibited (Fig. 2B, lane 2). Changing periods of hormone starvation from 3-7 days does not affect the basal level of TPO mRNA achieved (data not shown). Readdition of IGF-I (Fig. 2B, lanes 3 6), or TSH (Fig. 2B, lanes 7-10) for 48 h increases TPO mRNA levels, while it leaves unaltered the level of f3actin mRNA. The effect of both, IGF-I and TSH, is dose dependent. The mRNA levels are increased, respec-

B 1 2 3 4 5 6 7 8 9

28S-* 18S —

1

10

2

28S —

18S — Fig. 1. The Rat TPO cDNA Probe Identifies Tissue Specific mRNAs in the Rat Thyroid Northern blot analysis of TPO specific mRNA in different rat tissues and in FRTL-5 thyroid cells. A, RNA samples were applied to a 1 % agarose formaldehyde gel as follows: FRTL5 cells (lanes 1 and 2), thyroid (lanes 3 and 4), heart (lane 5), liver (lane 6), kidney (lane 7), brain (lane 8), testis (lane 9), and spleen (lane 10). Each lane contains 20 ng total RNA with the exception of lane 1 and 3 (10 ^g)- B, Poly (A)+ RNA of FRTL5 cells (lane 1) and of rat thyroid tissue (lane 2) was applied on 1.6 % agarose formaldehyde gel. Individual lanes contain 10 *tg (lane 1) and 2 ng (lane 2) poly (A)+ RNA, respectively. Mobility of 28S and 18S ribosomal RNA bands is indicated on the left of the figures. Rat /3-actin hybridization of the two blots has been used to analyze the amounts of RNA samples transferred on the filters (data not shown).

tively, 3- and 6-fold by IGF-I and TSH when normalized to rat /3-actin mRNA. The lowest effective concentrations are 0.01 (ig/m\ IGF-I and 1 x 10" 1 0 M TSH. The maximal effect is elicited at 0.1 ^g/ml IGF-I and 1 x 1 0" 8 M TSH. The same concentrations have been found to be effective on TG mRNA levels (Ref. 12 and Table 1)As shown in Fig. 3, the effect of IGF-I (Fig. 3B, lanes 2-7), as well as that of TSH (Fig. 3B, lanes 8-13), is time dependent: in both cases, using maximally effective doses, maximal induction is achieved 30 h after hormone addition and remains constant after 72 h and possibly longer. When the levels of TPO and TG mRNAs are compared (Table 1), it appears that they are increased by TSH and by IGF-I and or by insulin. In both cases, the effect of TSH is additive with IGF-I or insulin, while that of IGF-I is not additive with insulin. TSH has a larger effect on TPO mRNA levels than IGF-I, whereas TSH and IGF-I are equally effective in increasing TG mRNA levels. Since 5 /IM Forskolin is able to mimic TSH induction of both genes (Table 1), it is possible that the effect of TSH is cAMP mediated. r uptake in FRTL-5 cells shows a different regulation, being greatly increased (15-fold) by TSH, and not affected by insulin or IGF-I (Table 2). In the above experiments, we used the starvation conditions (0.2% serum and a four hormones mixture) reported by Santisteban et al. (12) to study the effect of TSH and IGF-l/insulin on TG synthesis in FRTL-5 cells. However, we cannot exclude that TSH and IGFl/insulin cooperate with other growth factors and hormones present in the starvation mixture. Modulation of Differentiated Functions in Normal and Transformed Rat Thyroid Cells In order to assess the TPO sensitivity to neoplastic transformation, we used normal PC cl.3 rat thyroid cells and PC cl.3 cells transformed with different oncogenes (13). This system has been already successfully employed to show a different sensitivity to transformation of TG synthesis and I" uptake (13). In the experiments discussed herein, normal PC cl.3 and FRTL-5 cells and PC myc, PC PyMLV, PC myc + PyMLV, and FRTL-5 KiMol transformed cells were chronically exposed to a mixture of six hormones, including TSH (3.7 x 1 0 - 8 M ) and insulin (1 ng/ml). As shown in Fig. 4, the two differentiated rat thyroid cell lines FRTL-5 (lanes 7 and 8) and PC cl.3 (lanes 1,2 and 9) exhibit similar expression of TG and TPO genes. TPO and TG mRNA levels are differentially affected by oncogene transformation of PC cl.3 cells. Thus, TPO and TG genes are still functioning in PC myc transformed cells (compare lanes 1 to 3, 10/ig total RNA, and lanes 2 to 4, 20 M g total RNA). However, densitometric analysis of the autoradiograph shows that TPO/actin and TG/actin mRNA levels are, respectively, 1.3 and 2-fold higher in PC myc cells compared to PC cl.3 cells. When PC cl.3 cells are transformed by PyMLV middle-T-antigen, TG gene is


41

TPO mRNA Levels in Rat Thyroid Cells

B

A

1 2 3 4 5 6 7 8 9

10

TPO §

ACT IN

Fig. 2. IGF-I and TSH Dose-Dependent Effect on TPO mRNA Levels in FRTL-5 Cells A, Ratio of TPO-reactive to /3-actin reactive gene transcripts in FRTL-5 cells exposed to different concentrations of TSH (upper curve, closed dots) and of IGF-I (lower curve, open triangles), as described in Materials and Methods. Messenger RNA values are expressed as fold increase over the basal level. The graph shows an average of densitometric scan values of at least three different experiments; the SD for each point was less than 10%. B, A representative Northern blot is shown. RNA samples (10 ng total RNA) were from: six hormones chronically stimulated cells (lane 1), cells grown without TSH, insulin and IGF-I (0.2% serum) (lane 2), IGF-l-treated cells (lanes 3-6), from the lower to the upper concentration of Fig. 2A), TSH-treated cells (lanes 7-10, from the lower to the upper concentration of Fig. 2A). Hybridizations of the same filter to TPO (top) or to /3-actin (bottom) probes are shown.

Table 1. Comparison of the Effect of TSH, IGF-I, Insulin, and Forskolin on the Steady State Levels of TPO and TG mRNAs Additions +TSH(10" 8 M) +IGF-I (0.1 Mg/ml) +lnsulin (1 Mg/ml) +TSH (10"8 M) + IGF-I (0.1 /ig/ml) +TSH (10~8 M) + Insulin (1 ^g/ml) +IGF-I (0.1 /xg/ml) + Insulin (1 fig/m\) +Forskolin (5 ^M) 1

TPO/Actin mRNA Levels" 5.9 3.1 2.6 9.0 8.2 4.0 5.6

± ± ± ± ± ± ±

0.5 0.4 0.2 0.8 0.6 0.3 0.2

TG/Actin mRNA Levels" 3.3 3.4 4.6 7.2 6.5 7.5 3.5

± ± ± ± ± ± ±

0.6 0.5 0.9 0.7 0.8 0.5 0.3

Messenger RNA values are expressed as fold increase over the basal level.

still expressed, while TPO gene is not expressed at all (lanes 5 and 6). Both genes appear to be switched off in PC cl.3 cells transformed by the cooperation of c-myc and PyMLV (lane 10) and in FRTL-5 KiMol transformed cells (data not shown). We analyzed, also, I" uptake in normal and transformed rat thyroid cells. Figure 5 shows a double reciprocal plot of I" uptake values vs. different I" concentrations (ranging from 5-200 /XM). The Michaelis-Menten constant (Km) estimated value (50 fiM) is similar in PC cl.3 and FRTL-5 cells chronically exposed to TSH and insulin and in PC cells transformed by c-myc oncogene or by polyoma middle-T-antigen grown in the same conditions (Fig. 5 and data not shown). This value is similar to that reported by Grollmann et al. (16) in FRTL5 cells and to that determined in rat thyroid slices (9)

and in primary culture (17). The maximum velocity (Vmax) values of I" uptake are different: thus, the PC myc cells take up r at a rate that is about 30% less that of normal PC cells, whereas the rate for polyoma transformed PC cells is about 75% less than that of normal PC cells (Table 3). Since the Km values of r uptake of normal PC and of PC myc and PC PyMLV cells are similar, the decrease in Vmax in transformed cells suggests a reduction in the number of active I" carrier molecules. I" uptake is totally absent in PC myc + PyMLV and FRTL-5 KiMol transformed cells (Table 3). DISCUSSION TSH is viewed as the important physiological regulator of the expression of the molecules involved in the


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MOL ENDO-1990 42

B 1 2 3 4 5 6 7 8 9 10 11 12 13 /

/

*

6

5

TPO

I s S Pi

/

/

3

2

ACTIN

1

i

•••••••§#»•••

II

Fig. 3. IGF-I and TSH Time-Dependent Effect on TPO mRNA Levels in FRTL-5 Cells A, Ratio of TPO-reactive to /3-actin reactive gene transcripts in FRTL-5 cells treated for different times with IGF-I (open triangles) or TSH {closed dots). Hormone concentrations were 0.1 Mg/ml for IGF-I and 10 ~8 M for TSH. Values plotted represent the average of three independent experiments; the SD for each point was less than 10%. B, A representative Northern blot is shown. RNA samples (10 ng total RNA) were from cells grown without insulin, IGF-I, and TSH (lane 1), IGF-l-treated (lanes 2-7) or TSH-treated (lanes 8-13) cells. Treatment times were from 5-72 h, as indicated. Hybridizations of the same filter to TPO (top) or to /3-actin (bottom) probes are shown.

Table 2. Effect of TSH, IGF-I, and Insulin on I" Uptake in FRTL-5 Cells Additions

I" Uptake (pmol/^g DNA)*

Basal (0.2% serum, no TSH, no IGF-I, no Insulin) +TSH(3.7x 10"8M) +IGF-I (0.001 +IGF-I (0.01 +IGF-I (0.1 -Hnsulin (0.01 +lnsulin (0.1 fig/m\) -Hnsulin (1 ng/m\)

0.8 ± 0.24 15.3 ±1.21 0.9 ± 0.38 0.6 ± 0.40 0.6 ± 0.33 1.0 ±0.24 0.7 ± 0.30 0.6 ± 0.43

a

FRTL-5 cells, routinely grown in a mixture of six hormones, were seeded at a density of 1 x 105/well in Falcon 24-wells culture dishes. After 48 h, cells were depleted to a basal state in a medium that contained no TSH, no insulin, no IGF-I (0.2% serum) for 5 days. The experiment was initiated by returning TSH, or insulin, or IGF-I at the concentrations indicated to the culture media for 48 h. I" uptake was measured at 5 min as described in Materials and Methods. The value of l~ uptake reported for each condition has been calculated by subtracting the value obtained in the presence of 100 HM NaCIO*. The results given are the mean ± SD of four experiments.

synthesis of thyroid hormones, i.e. TG (2-4) and TPO (4-8). TSH enhances, also, iodide trapping, although the molecular mechanism of such regulation is still unknown (4,9,10). Recently, it has been demonstrated, at least in the FRTL-5 cells, that also insulin and IGF-I, together with TSH, increase TG synthesis (12). We have investigated whether TPO expression and I" uptake also possess such a dual control by TSH and IGF-l/insulin in the FRTL-5 cells. We show that TPO mRNA levels are increased by either TSH or IGF-I additions. Both hormones exhibit the same time-course of induction with the maximal effect at 30 h. Recently, data obtained using primary

cultures of sheep (4) and dog (7) thyroid show that insulin does not influence TPO gene expression, whereas TSH does (4,7). This discrepancy with our data could be due to the different systems investigated. It is important to point out that, in FRTL-5 cells, TSH is more potent regulator of TPO mRNA levels than IGF-I/ insulin, while TSH and IGF-l/insulin are equally potent in increasing TG mRNA levels. Thus, our data confirm the difference in hormonal regulation between TPO and TG genes that has been already described in sheep and dog systems (4,7). We show that, in the FRTL-5 cells, I" uptake is not dependent at all on IGF-l/insulin, whereas it is strongly


43


1 2 3 4 5 6 7 8

9 10

TG

TPO

uptake induced by oncogene transformation is due, in fact, to a reduction of active I" carrier molecules. Our data, extending those previously reported by others (13,14) show that different thyroid specific functions exhibit different sensitivity to neoplastic transformation. Moreover, it is interesting to note that sensitivity of thyroid specific molecules to neoplastic transformation resembles the degree of their sensitivity to TSH regulation. In both cases, the inhibition of the expression of these molecules follows the same hierarchy: the most sensitive molecule is I" carrier, the less sensitive is TG. Although the repression of tissue specific functions that occurs in transformed thyroid cells is mediated by a complex mechanism, our data would suggest that part of this phenomenon is due to the interference of oncogene transformation with the TSH action on thyroid cells.

ACTIN Fig. 4. Modulation of TPO and TG mRNA Expression in Normal and Transformed Rat Thyroid Cells Northern blot analysis of TG, TPO, and /3-actin mRNAs of the following rat thyroid cell lines: FRTL-5 (lanes 7-8), PC cl.3 (lanes 1-2, 9), PC myc (lanes 3-4), PC PyMLV (lanes 5-6), PC myc + PyMLV (lane 10). Normal and transformed thyroid cells were chronically exposed to a mixture of six hormones including TSH (3.7x10 ~8 M) and insulin (1 fig/m\). Ten micrograms (lanes 1, 3, 5, 7) and 20 ^g (lanes 2, 4, 6, 8, 9, 10) of total RNA were loaded for each different cell line analyzed, except for PC myc + PyMLV (lane 10), where 20 ng were loaded. The same filter was sequentially hybridized to the different probes shown in the figure.

affected (15-fold) by TSH. Similar data have been reported in primary cultures of sheep thyroid cells (4). Neoplastic transformation interferes with the differentiated phenotype of thyroid cells (13,14). In FRTL-5 cells, TG expression and I" uptake are abolished after Ki-ras oncogene transformation (14,18,19). In the PC cl.3 cells, a differential inhibition of TG synthesis and I" uptake is observed after transformation by c-myc or PyMLV (13). c-myc or PyMLV separately, reduced I" uptake, but not TG expression, while cooperation of cmyc and PyMLV inactivates both differentiated functions (13). In the present study, we have analyzed TPO sensitivity to oncogene transformation in PC cells. We demonstrate that in the PC myc transformed cells, I" uptake is slightly decreased, while TPO and TG mRNA levels are not; in the PC PyMLV transformed cells, TPO expression is not detectable, I" uptake is greatly inhibited and only the TG gene is normally expressed. All three thyroid functions are abolished in transformed thyroid cells displaying a more malignant phenotype (PC myc + PyMLV transformed cells). The transformation sensitivity of I" uptake reported in this study is similar to that previously described (13). Moreover, we determined that the repression of I"

MATERIALS AND METHODS Reagents Purified TSH from Sigma (St. Louis, MO); bovine insulin from Calbiochem (La Jolla, CA); rat IGF-I from AMGem Biologicals. Guanidine isothiocyanate was purchased from Fluka. a32 PdGTP (800 Ci/mmol) and a32 PdATP (800 Ci/mmol) were from Dupont NEN Research Laboratories (Boston, MA). Restriction endonucleases used were purchased from Boehringher Indianapolis, IN). The random priming labeling reagents were purchased from Amersham. Cell Lines and Tissues A group of 10 normal male Wistar rats (250 g in weight) were used as sources of tissues fir RNA preparation. The isolation, growth, and properties of FRTL-5, FRTL-5 KiMOL, and PC cells have been described elsewhere (11,13,14). The FRTL-5 cells were routinely grown in Coon's modified Ham's F-12 medium supplemented with 5% calf serum (GIBCO, Grand Island, NY) and a six hormones mixture including TSH (3.7 x 10"8 M) and insulin (1 M9/ml). The PC cl.3, PC myc, PC PyMLV, PC myc + PyMLV, and FRTL-5 KiMOL transformed cells were grown in the same media conditions used for the FRTL-5 cells. When the effect of the hormones on TPO and TG expression was studied, FRTL-5 cells were plated in 100-mm tissue culture dishes at a density of 1.5 x 106 cells per dish. After 48 h, cells were starved in a medium containing 0.2 % calf serum, and a four hormones mixture without TSH and insulin. After 5 days starvation, TSH, insulin, and/or IGF-I were added to the culture medium at the concentrations noted. After another 48 h (unless otherwise noted) the cells were harvested for RNA extraction. Hybridization Probes The rat TPO cDNA probe we used was a generous gift from Dr. R. Di Lauro (EMBL, Heidelberg, West Germany). It was isolated by screening a rat thyroid lambda gtll cDNA library with a porcine TPO probe (20) (Di Lauro et a/., unpublished results). A 0.7 kb £coRI-Sacl fragment corresponding to the 5'-coding region of the rat cDNA was purified, labeled and used as a probe in the hybridization experiments. TG cDNA probe used was a 680 base pair £coRI-Psfl fragments corresponding to the 3' end of the rat TG cDNA (21). /3-actin probe used was a 2.0 kb SamHI-Sam-HI insert isolated from a rat B-


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Fig. 5. The Effect of External I Concentration on l~ Uptake into PC cl.3 (x), PC myc (A), and PC PyMLV (a) Cells Cells routinely grown in a mixture of six hormones, were seeded at a density of 1 X 1 0 5 cells per well in Falcon 24-wells tissue culture dishes. The cells were used in individual experiments 3 days after plating. I" uptake was determined as detailed in Materials and Methods. The reactions were terminated at 5 min, as described. The value of r uptake reported for each condition has been calculated by subtracting the value obtained in the presence of 100 ^M NaCI04. Each point is the mean ± SD of at least six experiments. vmax and Km values were obtained using a computer program.

Table 3. Modulation of Differentiated Thyroid Functions in Normal and Transformed Thyroid Cell Lines Cell Type

Vmax of r Uptake (pmol/^g DNA/min)"

PC cl.3 PC myc PC PyMLV PC myc + PyMLV FRTL-5 FRTL-5 KiMol

6.7 4.5 1.6 14.3 _

TPO mRNA

TG mRNA

a

vmajt values are calculated from data of Fig. 5.1" uptake data reported herein are similar to those obtained by Fusco et al. (13). Quantitative discrepancies between our data and those previously reported (13,14), are probably due to the different method of I" uptake measurement we used.

actin cDNA plasmid kindly provided by Dr. B. Patterson (NIH, National Cancer Institute). RNA Isolation Cells were washed twice with cold PBS, treated with 4 M guanidine isothiocyanate, and removed from plates with a teflon policeman. Rat tissues were frozen in liquid nitrogen immediately after the animal was killed. Frozen tissues were homgenized in 4 M guanidine isothiocyanate by a polytron

mixer. Total and poly(A)+ RNAs were prepared using standard procedures (22-24). Northern Blot Analysis RNA samples were electrophoresed through a 2.2 M formaldehyde agarose gel (24), and transferred to Nytran membranes (Scheicher and Schuell, Keene, NH). Filters were hybridized to a «32P random primed probe (25) with a specific activity of 1 x10 9 cpm/^g DNA, according to the manufacturers protocol (Schleicher and Schuell). Filters were washed with two changes of 2x SSPE and 0.1% (sodium dodecyl sulfate) at room temperature for 15 min each time and in the same solution at 50 °C for 30 min. The final wash was carried out in 0.2x SSPE for 30 min. 55 °C. Filters were exposed to Kodak XAR-5 film at - 7 0 °C with intensifying screening. TPO, TG, or /3-actin mRNA levels were quantified by densitometric scanning of the autoradiographs using an LKB Ultrascan densitometer. The values were expressed as the ratio of TPO//3actin or TG/j8-actin reactive hybridization values measured in the densitometer under the same conditions. Each point represents the average of several independent determinations. I" Uptake Measurements Culture medium was aspirated and the cells were washed with 1 ml buffered Hanks buffered salt solution (HBSS) pH 7.3 (10). r uptake was initiated by adding 0.5 ml HBSS containing either 0.1 /iCi carrier free Na125l and 10 HM Nal and carrier-free Na125l to give a constant specific activity of 50 mCi/mmol. Incubations proceeded at 37 °C in a humidified atmosphere



for 5 min and were terminated by aspirating the radioactive medium and washing the dishes with 1 ml/well ice-cold HBSS. To determine the amount of 12SI associated with the cells, 1 ml 95% ethanol was added to each well for 20 min and then transferred into vials for counting with a Beckman 5500 ycounter. I" uptake values were expressed as pmoles per ^g DNA. The DNA was determined for each well on the material not extracted by ethanol, as previously reported (26). DNA values ranged from 4-8 M9/well for the individual experiments described in this report.

Acknowledgments We thank Dr. Roberto Di Lauro (EMBL, Heidelberg, West Germany), who kindly provided the rat TPO probe; Dr. Alfredo Fusco, for the PC cl.3, PC myc, PC PyMLV, and PC myc + PyMLV cell lines. We are also grateful to Dr. Enrico Avvedimento for stimulating discussions and to Drs. Salvatore Maria Aloj, Carmelo Bruno Bruni, Stella Carlomagno, and Valeria Ursini for critical reading of the manuscript.

Received August 10, 1989. Revision received September 29,1989. Accepted September 29,1989. Address requests for reprints to: Dr. Raffaele Zarrilli, Dipartimento di Biologia e Patologia Cellulare e Molecolare, II Facolta di Medicine e Chirurgia, Via S. Pansini, 5,80131, Napoli, Italy.

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Sterol regulatory element-binding proteins are regulators of the rat thyroid peroxidase gene in thyroid cells.

Demonstration of the existence of the alternatively spliced form of thyroid peroxidase in normal thyroid.

Improved assay procedures for thyroid peroxidase; application to normal and adenomatous human thyroid tissue.

Thyroid peroxidase, thyroglobulin, cAMP and DNA in human thyroid.

Thyroid peroxidase and the induction of autoimmune thyroid disease.

Hormonal regulation of major histocompatibility complex class I genes in rat thyroid FRTL-5 cells: thyroid-stimulating hormone induces a cAMP-mediated decrease in class I expression.

Growth regulation of normal thyroids and thyroid tumors in man.

Studies with purified human thyroid peroxidase and thyroid microsomal autoantibodies.

Regulation of differentiation in normal and transformed erythroid cells.

Organization of tubulin in normal and transformed rat kidney cells.

Thyroid peroxidase activity in iodine deficient rats.

Thyroid peroxidase in endemic goiter tissue.

Effects of retinoids on iodine metabolism, thyroid peroxidase gene expression, and deoxyribonucleic acid synthesis in porcine thyroid cells in culture.

Antithyroid peroxidase autoantibodies in thyroid diseases.

Thyroid peroxidase glycosylation: the location and nature of the N-linked oligosaccharide units in porcine thyroid peroxidase.

Iodothyronine-induced catalatic activity of thyroid peroxidase.

Methimazole regulation of thyroglobulin biosynthesis and gene transcription in rat FRTL-5 thyroid cells.

The molecular biology of thyroid peroxidase: cloning, expression and role as autoantigen in autoimmune thyroid disease.

Antigen-specific T cell recognition of affinity-purified and recombinant thyroid peroxidase in autoimmune thyroid disease.

The mRNA levels of thyrotropin receptor, thyroglobulin and thyroid peroxidase in neoplastic human thyroid tissues.

Value of thyroid specific peroxidase and Ki-67 stains in preoperative cytology for thyroid follicular tumors.

Male gender differences in the thyroid ultrasound features, thyroid peroxidase antibodies and thyroid hormone levels: a large population-based study.

Regulation of thyroid hormone synthesis in cultured ovine thyroid follicles.

Heterogeneity of autoantibodies against thyroid peroxidase in autoimmune thyroid disease: evidence against antibodies directly inhibiting peroxidase activity as regulatory factors in thyroid hormone metabolism.