Defective Mitochondrial ATP Synthesis in Oxyphilic Thyroid Tumors
                    F. Savagner, B. Franc, S. Guyetant, ...
0013-7227/01/$03.00/0                                                           The Journal of Clinical Endocrinology & Me...
Savagner et al. • ATP Synthesis in Oxyphilic Thyroid Tumors                               J Clin Endocrinol Metab, October...
4922   J Clin Endocrinol Metab, October 2001, 86(10):4920 – 4925                                Savagner et al. • ATP Synt...
Savagner et al. • ATP Synthesis in Oxyphilic Thyroid Tumors                     J Clin Endocrinol Metab, October 2001, 86(...
4924   J Clin Endocrinol Metab, October 2001, 86(10):4920 – 4925                            Savagner et al. • ATP Synthesi...
Savagner et al. • ATP Synthesis in Oxyphilic Thyroid Tumors                                     J Clin Endocrinol Metab, O...
Upcoming SlideShare
Loading in …5

Defective Mitochondrial ATP Synthesis in Oxyphilic Thyroid Tumors


Published on

Published in: Health & Medicine
  • Be the first to comment

  • Be the first to like this

No Downloads
Total views
On SlideShare
From Embeds
Number of Embeds
Embeds 0
No embeds

No notes for slide

Defective Mitochondrial ATP Synthesis in Oxyphilic Thyroid Tumors

  1. 1. Defective Mitochondrial ATP Synthesis in Oxyphilic Thyroid Tumors F. Savagner, B. Franc, S. Guyetant, P. Rodien, P. Reynier and Y. Malthiery J. Clin. Endocrinol. Metab. 2001 86: 4920-4925, doi: 10.1210/jc.86.10.4920 To subscribe to Journal of Clinical Endocrinology & Metabolism or any of the other journals published by The Endocrine Society please go to: Copyright © The Endocrine Society. All rights reserved. Print ISSN: 0021-972X. Online
  2. 2. 0013-7227/01/$03.00/0 The Journal of Clinical Endocrinology & Metabolism 86(10):4920 – 4925 Printed in U.S.A. Copyright © 2001 by The Endocrine Society Defective Mitochondrial ATP Synthesis in Oxyphilic Thyroid Tumors F. SAVAGNER, B. FRANC, S. GUYETANT, P. RODIEN, P. REYNIER, AND Y. MALTHIERY Inserm EMI-U 00-18 (F.S., P.R., P.R., Y.M.), Laboratoire de Biochimie et Biologie Moleculaire, Angers F-49033; Laboratoire ´ d’Anatomie Pathologique (B.F.), Hopital Ambroise Pare, Boulogne F-92104; Laboratoire d’Anatomie Pathologique (S.G.); ˆ ´ and Service d’Endocrinologie (P.R.), Nutrition et Medecine Interne, Angers F-49033, France ´ Oxyphilic tumors (oncocytomas or Hurthle cell tumors) form ¨ oxygen in mitochondria from tumors was determined by po- a rare subgroup of thyroid tumors characterized by cells con- larography. ATP assays were used to explore the mitochon- taining abundant mitochondria. The relationship between drial respiratory chain activity and the oxidative phosphor- the mitochondrial proliferation and the pathogenesis of these ylation coupling in seven fresh thyroid tumors and controls. tumors is unknown. We have assessed the expression of the Adenosine triphosphate synthesis was significantly lower in mitochondrial ND2 and ND5 (subunits of the nicotinamide all the tumors, compared with controls, suggesting that a cou- adenine dinucleotide dehydrogenase complex) genes and the pling defect in oxidative phosphorylation may be a cause of nuclear UCP2 (uncoupling protein 2) gene in 22 oxyphilic mitochondrial hyperplasia in oxyphilic thyroid tumors. (J thyroid tumors and matched controls. The consumption of Clin Endocrinol Metab 86: 4920 – 4925, 2001) O XYPHILIC THYROID TUMORS, also known as onco- cytomas or Hurthle cell tumors, represent a rare sub- ¨ group of follicular thyroid neoplasms, characterized by cells tissue and muscle/white fat tissues, respectively (10). How- ever, the histochemical investigation of the key nuclear com- ponents involved in the oxidative phosphorylation process with a distinctive eosinophilic cytoplasm (1). The cytoplas- has revealed no coupling defects in oxyphilic thyroid tumors mic eosinophilia is owing to the abundance of morpholog- (11). ically altered mitochondria in the majority of tumor cells (1, The proliferation of mitochondria in oxyphilic tumors 2). However, the relationship between mitochondrial prolif- might result from the induction of genes involved in mito- eration and the histogenesis of oxyphilic tumors is unknown. chondrial biogenesis. The patterns of mitochondrial tran- The development of mitochondria involves the synthesis scripts (especially ND2 and ND5) as well as nuclear tran- of proteins encoded by mitochondrial DNA (mtDNA), which scripts from certain genes coding for proteins involved in carries the genes for the essential subunits of the respiratory oxidative phosphorylation differ according to whether the chain complexes, as well as by nuclear DNA (nDNA). Elec- tumor is a renal oxyphilic tumor or a salivary gland oxyphilic trons from the nicotinamide adenine dinucleotide generated tumor (12). Changes in mtDNA transcription and mitochon- by glycolysis in the cell are transported into the mitochondria drial mRNA stability were observed in the former but not in in which the flow of electrons between the respiratory chain the latter, suggesting that the process of mitochondrial pro- complexes supplies the energy used by ATP synthase to liferation varies according to the origin of the tumor. produce ATP. An alternative source of energy, independent We examined thyroidectomy specimens from 22 anony- of ATP synthase, is provided by the uncoupling protein mous patients. In each case, tissue removed from the normal (UCP), which plays an important role in energy homeostasis part of the thyroid gland served as a control for the oxyphilic (3). The analysis of mtDNA in several oxyphilic tumors has thyroid tumor. For each set of paired specimens, we deter- shown that the abnormally high histochemical activity of the mined the gene expression profiles of the mitochondrial ND2 respiratory chain complexes is associated with a great in- and ND5 genes as well as the nuclear UCP2 gene involved crease in the amount of wild-type mtDNA (4). in energy production. Fresh tissue samples, obtained from 7 The frequency of aneuploid or polyploid cells in oxyphilic of the 22 patients, were analyzed by polarography to inves- thyroid tumors suggests the presence of anomalies in nuclear tigate the mitochondrial respiratory chain activity, the rate of genes (5). Mitochondrial proliferation has also been reported oxidative phosphorylation (ADP/oxidation ratio) and ATP in mitochondrial diseases associated with respiratory chain assays were used to determine the mitochondrial ATP defects or coupling defects between the respiratory chain and synthesis. ATP production (6 –9). Among the UCPs that induce ther- mogenesis during mitochondrial respiration (3), UCP2 is the Materials and Methods unique form expressed in many tissues and cell types. In particular, UCP2 is expressed in thyroid tissue, whereas the Thyroid tissue samples expression of UCP1 and UCP3 is limited to brown fat adipose Twenty-two benign or malignant oxyphilic thyroid tumors, diag- nosed between 1992 and 2000 at the Ambroise Pare Hospital, Paris (15 ´ cases) and the University Hospital, Angers (7 cases), were included in Abbreviations: mtDNA, Mitochondrial DNA; nDNA, nuclear DNA; the study. All the samples used were rendered anonymous (i.e. all rDNA, ribosomal DNA; UCP, uncoupling protein. patient identifiers were deleted before the study). The cases were con- 4920
  3. 3. Savagner et al. • ATP Synthesis in Oxyphilic Thyroid Tumors J Clin Endocrinol Metab, October 2001, 86(10):4920 – 4925 4921 secutive and unselected apart from exclusions on account of insufficient milligram of mitochondrial protein) was determined for intact and per- material or association of the tumors with chronic thyroiditis. Nineteen meabilized mitochondria. of the tumors were follicular oxyphilic adenomas (six of which were trabecular) and three were follicular oxyphilic carcinomas. Five of the DNA isolation and Southern blot analysis adenomas were associated with a multinodular goiter. The diagnoses were made according to the World Health Organization classification DNA was isolated using the phenol-chloroform procedure. The sam- (1). Oxyphilic adenoma was distinguished from carcinoma on the basis ples were digested overnight with RNase A (20 g/ml) and proteinase of vascular or capsular invasion or metastasis. The patients were 2 men K (20 mg/ml) at 37 C in Tris-HCl 10 mm, EDTA (pH 8) 0.1 m, and SDS and 20 women, with a mean age of 53 yr (range 27– 82 yr). 0.5%. The proteins were removed by organic extraction followed by The average size of the tumor was 37.8 20.5 mm (mean sd; range ethanol precipitation with NaCl 0.2 m and centrifugation for 15 min at 15–90 mm). In addition to neoplastic thyroid samples, normal thyroid 10,000 g. Five micrograms of DNA were digested with the restriction samples were taken sufficiently distant from the tumors to serve as enzyme XbaI (Biolabs, Beverly, MA). Southern blotting was performed controls. All the samples were immediately stored in liquid nitrogen according to standard methods. Probes labeled by digoxigenin were until extraction of high-molecular-weight DNA and RNA. obtained by multirandom priming and were revealed with antidigoxi- The 22 tumor samples and controls were fixed in formalin, embedded genin antibodies labeled by alkaline phosphatase (DigDNA labeling and in paraffin, and stained with hematoxylin and eosin. Immunohisto- detection kit, Roche, Basel, Switzerland). The mitochondrial and nDNA chemistry was performed on paraffin-embedded sections. Tissue sam- were detected by using the probes 12S ribosomal DNA (rDNA) (nt ples from the seven fresh tumors (six adenomas and one carcinoma) and 592-1344) and 18S rDNA (nt 1201–1811), respectively. For each sample, matched controls were kept in a preservative medium [100 mm sucrose, the intensities of the mtDNA signals, and the corresponding nDNA 1 mm EGTA, 20 mm 3-[N-morpholino]propanesulfonic acid (pH 7.4), 1 signals, were quantified by densitometric analysis (Molecular Analyst, g/liter bovine albumin] to prepare the mitochondria for polarographic Bio-Rad Laboratories, Inc., Cambridge, MA). studies and ATP measurement. Tumoral and control tissues were compared using a nonparametric RNA isolation and cDNA synthesis test for matched-pair samples (Wilcoxon test), the differences being considered statistically significant at P less than or equal to 0.05. All the RNA was isolated using the guanidinium isothiocyanate procedure numerical values below are expressed as means sd. (Trizol Reagent, Life Technologies, Inc., Gaithersburg, MD). Residual DNA was removed by DNase treatment: 5 g total RNA were incubated with 2 U RNase-free DNase I for 1 h at 37 C. Immunohistochemistry To generate cDNA, 1 g of RNA was first denatured at 70 C with 1 After morphological examination of hematoxylin- and eosin-stained m of oligodT (Promega Corp.) for 5 min before quenching on ice; then sections, corresponding 3- m sections of the paraffin blocks were pre- 0.5 mm of each of the 4 dNTPs, 10 mm dithiothreitol, 10 U RNase pared for the detection of mitochondrial antigen expression as a semi- inhibitor, and 200 U superscript II (Life Technologies, Inc.) were added quantitative index of mitochondrial biogenesis. A monoclonal antibody to the 5 buffer to make up a final volume of 20 l reaction mix. The 113–1 was used, recognizing an unknown 60-kDa nonglycosylated pro- reaction mix was incubated for 1 h at 42 C. The reverse transcriptase was tein component of human cell mitochondria (BioGenex Laboratories, inactivated at 70 C for 15 min. Inc., San Ramon, CA). Immunostaining was performed with the stan- dard avidin-biotin peroxidase technique with antigen retrieval. For neg- Quantitative PCR analysis ative control slides, the primary antibody was either omitted or replaced by a suitable concentration of normal IgG of the same species. Real-time quantitative PCR with an external standard was used to determine the gene copy number (Lightcycler, Roche). Standard PCR products for each gene were generated by amplifying nuclear cDNA or Polarographic studies mtDNA templates. PCR products were purified by the phenol-chloro- form method and the copy number in the final sample was determined Oxygen consumption was measured with a Clark electrode at 30 C by two independent methods (i.e. spectrophotometry and gel analysis). in a 2-mL chamber (Oxygraph OROBOROS, Anton Paar, Innsbruck, ¨ For each gene tested, a sequence-specific standard curve was plotted Austria). The chamber was isolated from contact with the atmosphere using serial dilutions of the target gene standard PCR product, and the by a close-fitting cap so that the electrode current was proportional to same primers were used to amplify the cDNA. the partial pressure of oxygen in the sample. Substrates for the different The expression of two mitochondrial genes, ND2 and ND5, and two complexes of the respiratory chain were introduced into the chamber nuclear genes, UCP2 and -ACTIN, was analyzed using the PCR primer and consumed by mitochondria in the oxyphilic tumors or matched sets indicated in Table 1. The amount of RNA determined for each controls. Mitochondria were isolated from seven fresh tissue samples sample was normalized by the quantification of the -ACTIN transcripts. and matched controls using the standard procedure (13). Oxygen uptake Two microliters of master mix containing Taq DNA polymerase, was measured after the addition of mitochondria (200 g protein) to 2 dNTPs, and SYBR green I (DNA Master SYBR Green I kit, Roche) were ml incubation medium [300 mm mannitol, 10 mm KH2PO4 (pH 7.2), 10 incubated for 5 min at room temperature with 0.16 l of Taqstart an- mm KCl, and 5 mm MgCl2] to determine the basal respiratory activity. tibody. The PCR reaction was then started by adding MgCl2 4 mm and Substrates and inhibitors were introduced into the oxygraph chamber forward and reverse primers (0.5 m) to the capillary tubes of the through the stopper port. First, 2 l EDTA (20 mm) and 2 l rotenone Lightcycler apparatus containing the master mix and 2 L of template (5 mm) were added to inhibit exogenous ATPase and complex I (nico- (cDNA or a standard with a known copy number) in a final volume of tinamide adenine dinucleotide ubiquinone oxidoreductase of the respi- 20 l. ratory chain), respectively. Then, 20 l succinate (1 m) and 10 l ADP (30 mm) were successively added to the sample to determine the rate of oxidative phosphorylation (ADP/O ratio). Finally, 10 l potassium cy- anide (200 mm) were added to stop oxygen uptake by inhibiting complex TABLE 1. Oligonucleotide pairs used for quantitative PCR IV (cytochrome c oxidase of the respiratory chain). Primer pairs Genes 5 -GCACCCCTCTGACATCC-3 ND2 ATP measurement 5 -CGGTCGGCGAACATCAGTGG-3 Mitochondrial ATP was measured by bioluminescence using the 5 -GGGGATTGTGCGGTGTGTG-3 ND5 luciferin-luciferase reaction (Enliten, Promega Corp., Madison, WI) (14). 5 -CTTCTCCTATTTATGGGGGT-3 Mitochondria were isolated, using the same methods as for the polaro- 5 -CCAGTGCGCGCGCTACAGTCA-3 UCP2 graphic studies, from the seven fresh oxyphilic tumor samples and their 5 -GTGGTGCTGCCTGCTAGGAG-3 matched controls. After incubation of mitochondria with 10 mm gluta- 5 -CGACATGGAGAAAATCTGGC-3 -ACTIN mate and malate for 10 min, the rate of ATP synthesis (expressed per 5 -AGGTCCAAGACGCAGGATGG-3
  4. 4. 4922 J Clin Endocrinol Metab, October 2001, 86(10):4920 – 4925 Savagner et al. • ATP Synthesis in Oxyphilic Thyroid Tumors Results mitochondria, attesting to the functionality of ATP/ADP Immunohistochemistry translocase in exporting ATP from intact mitochondria. The 60-kDa mitochondrial protein was present in the cy- MtDNA quantification toplasm of all the 22 oxyphilic thyroid tumor samples, both homogeneous (19 tumors) and heterogeneous (3 tumors) Twenty-two tumors and matched controls were explored distributed (histochemistry score data not shown), confirm- for mtDNA quantification. DNA was preserved from rRNA ing the increased mitochondrial biogenesis in the tumor sam- contamination by buffered RNase during extraction. The ples. In contrast, the immunostaining of the matched control ratio of mtDNA to nDNA in oxyphilic tumor samples and samples was extremely weak or undetectable. The homoge- matched controls was determined by Southern blot analysis. neous distribution of mitochondrial immunostaining with Three hybridization bands were detected by the mitochon- no difference between adenomas vs. carcinomas has been drial 12S rRNA probe (7.5 and 1.7 kb) and the nuclear 18S previously described for oxyphilic tumors (15). rRNA probe (1.5 kb). The densitometric analysis of the 1.7- and 1.5-kb bands showed that the 12S rDNA/18S rDNA ratio Polarographic analysis was 1.67 0.09 in oxyphilic thyroid tumors, compared with 0.54 0.19 in controls. This increase in mtDNA content for Seven tumors and matched controls (n 14) were used for tumors was highly significant, using the Wilcoxon test (P the functional analysis of the respiratory chain. The mito- 0.001). Fig. 1 shows the 1.7- and 1.5-kb bands of several chondria were prepared within 1 h after thyroidectomy so as oxyphilic tumors samples and matched controls. to obtain interpretable results. Polarographic analysis of Deletions in mtDNA were explored by long PCR analysis complexes I to IV produced no evidence of respiratory chain using a standard procedure (16). The common mtDNA4977 defects in the seven oxyphilic tumors vs. control tissue. The deletion was found in two of the tumors as well as in the respiratory control indices were calculated by dividing the matched controls, with an identical level of heteroplasmy. rate of oxygen uptake in state 3 (stimulated by ADP addition) All the other samples were free from this deletion. by the rate of oxygen uptake in state 4 (when all the ADP is converted to ATP). Using succinate as substrate, the indices Mitochondrial and nuclear gene expression were 3.8 0.8 for tumors and controls (n 14). The ADP/O ratio in the tumor samples was 1.2 0.3 (n 7), whereas it Twenty-two tumors and matched controls were explored was 1.6 0.4 in the matched controls (n 7), but this for mtDNA quantification. The expression of ND2 and ND5 difference is not significant (Table 2). mitochondrial genes was 12 times higher in oxyphilic thyroid tumor samples than in controls. When adjusted to the ATP synthesis mtDNA/nDNA ratio, the relative mitochondrial transcript Because the analysis of ATP synthesis can be usefully ratio was 3.8 times higher in the tumor samples than in performed exclusively on mitochondria from fresh tissues, controls. only the mitochondria from the seven tumors and their For the nuclear gene, we observed a 2-fold increase of matched controls (n 14), as used for the polarographic UCP2 in oxyphilic tumors samples, compared with controls. studies, were used for ATP measurement. The mitochondrial Fig. 2 shows the different patterns of mitochondrial and ATP synthesis, adjusted to the mitochondrial protein level nuclear gene expression in the tumor samples and controls. after addition of the substrate, was 5.8 1.4 mol/mg per Table 3 summarizes the histology and the gene expression 10 min in oxyphilic tumors samples, compared with 12.1 pattern of the different samples. Table 2 sums up the statis- 3.1 mol/mg per 10 min in matched controls. This represents tical analysis of the results. a low rate of mitochondrial ATP synthesis in comparison Discussion with the basal rate for normal thyroid tissue we had previ- ously measured (13.5 2.8) (unpublished data). The Wil- Several authors have suggested that defective energy- coxon test showed that the decrease observed in ATP syn- producing mechanisms of oxyphilic cells may be responsible thesis was highly significant (P 0.018, Table 2). There was for mitochondrial proliferation (11, 17). This hypothesis no difference in ATP levels between intact and permeabilized stems from the observation that the active metabolism of TABLE 2. Statistical analysis of mitochondrial (ND2, ND5) and nuclear (UCP2) gene expression and ATP synthesis in oxyphilic thyroid tumors, compared with matched controls Control tissuea Oxyphilic tumorsa Wilcoxon test (n 22) (n 22) ND2b 658 290 6799 3215 P 0.001 ND5b 612 302 7354 3208 P 0.001 UCP2b 272 119 503 191 P 0.001 n 7 n 7 ADP/O ratio 1.6 0.4 1.2 0.3 NS ATP ( mol/mg protein per 10 min) 12.1 3.1 5.8 1.4 P 0.018 a Mean SD. b Copy number/ -ACTIN copy number. ADP/O ratio, Rate of oxydative phosphorylation; NS, not significant.
  5. 5. Savagner et al. • ATP Synthesis in Oxyphilic Thyroid Tumors J Clin Endocrinol Metab, October 2001, 86(10):4920 – 4925 4923 FIG. 1. Quantitation of mtDNA and nDNA for several oxyphilic thyroid tu- mors and matched controls by Southern blot analysis. Five micrograms of total DNA digested with XbaI was hybrid- ized first with an 18S rRNA nuclear probe and then rinsed and hybridized with a 12S rRNA mtDNA probe. The intensities of the 1.5-kb (18S) and 1.7-kb (12S) bands were quantified by a RadioImager (Cyclone, Packard, Down- ers Grove, IL). C, Control tissue; O, oxy- philic thyroid tumor. any mtDNA deletions that might provide a replicative advan- tage over wild-type mtDNA. The “common” mtDNA4977 de- letion was found in 2 of the 22 oxyphilic tumors investigated, but in each case the same deletion was also detected in the corresponding controls and corresponded to two elderly pa- tients (63 and 75 yr). Thus, the mtDNA4977 deletion might be associated with cellular aging rather than to the development of an oncocytic phenotype, as previously suggested (21, 22). The increase in mtDNA content observed in 22 thyroid tumors (3.10 0.29) was 25% lower than that indicated by other authors (4.31 1.09) (21). The analysis of mitochondrial gene expression showed that tumors had a 12-fold increase in ND2 and ND5 tran- scripts, compared with control tissue. The expression of these two mitochondrial genes has already been associated with abnormal mitochondrial biogenesis in oncocytic tumors (12). The gene expression ratio was adjusted to the mtDNA/ nDNA ratio calculated by Southern blot analysis to obtain a more accurate estimate of the real increase of mitochondrial gene expression. The mtRNA/mtDNA ratio thus deter- mined for the oxyphilic thyroid tumors may be compared with that given for oxyphilic tumors in other tissues. We found an mtRNA/mtDNA ratio of about 4:1 in oxyphilic thyroid tumors, compared with controls, whereas this ratio was 1:1 in the case of oxyphilic salivary gland tumors and 1:5 in the case of oxyphilic renal tumors (12). In the study of a FIG. 2. Quantification of mitochondrial and nuclear gene transcripts cell line derived from a thyroid oncocytoma, we found an from 22 oxyphilic thyroid tumors and matched controls. 2a, Mean ( SD) of the ratio of the mitochondrial ND2 and ND5 cDNA copy num- mtRNA/mtDNA ratio as high as 2:1, compared with a con- bers vs. the -ACTIN cDNA copy number. 2b, Mean ( SD) of the ratio trol thyroid cell line (23). These large differences in the of the nuclear UCP2 cDNA copy number vs. the -ACTIN cDNA copy mtRNA/mtDNA ratio suggest tissue-specific regulation of number. The cDNA copies were determined by real-time quantitative mitochondrial transcription and replication. It might there- PCR analysis (Lightcycler, Roche, Basel, Switzerland) after reverse transcription of the total RNA of each tumor and control sample. fore be relevant to investigate the nuclear factors involved in (Table 1 shows the PCR primers used.) this regulation in various tissues. Polarographic analysis produced no evidence of respi- oncocytic cells, with their high levels of oxidative enzymes, ratory chain defects in oxyphilic thyroid tumors, com- does not correspond to high thyroid cell function (18). How- pared with control tissue. The respiratory chain ratios in ever, in these histochemical studies, the respiratory enzymes mitochondria isolated from seven oxyphilic tumors were were functional, and a protein uncoupling the oxidative consistent with the indices published for mitochondria in phosphorylation process (UCP1) was not present in oxyph- the normal thyroid (24). However, the ADP/O ratio was ilic tumors (11). only 75% of the normal value. The oxidative phosphory- MtDNA alterations are associated with several mitochon- lation coupling defect revealed by polarography might be drial degenerative diseases (19). Large mtDNA deletions, related to the 2-fold increase in UCP2 expression observed such as the most common mtDNA4977 deletion, result in a in oxyphilic tumors, compared with controls. After veri- decline of the oxidative phosphorylation capacity and accu- fying that UCP1 was not expressed in oxyphilic thyroid mulate progressively in aging normal tissues (20). An in- tumors (data not shown), we investigated the expression creased frequency of the mtDNA4977 deletion in oncocytic of UCP2, the role of which has been established in the tumors has been described (18). We were unable to identify uncoupling process (25).
  6. 6. 4924 J Clin Endocrinol Metab, October 2001, 86(10):4920 – 4925 Savagner et al. • ATP Synthesis in Oxyphilic Thyroid Tumors TABLE 3. Case reports ND2# ND5# UCP2# Case Histology C O C O C O 1 OTA 752 7,890 812 6,850 355 469 2 OFA 267 5,749 320 7,760 123 198 3 OFC 402 2,592 130 1,390 420 972 4 OFA 1,140 14,587 1,250 11,760 201 495 5 OFA 760 8,403 548 11,540 341 507 6 OFC 804 8,702 647 12,501 201 357 7 OFA 589 3,015 950 5,470 258 398 8 OTA 549 4,057 450 5,690 507 980 9 OFA 705 9,587 521 12,589 204 480 10 OFA 941 5,783 203 2,547 438 508 11 OTA 875 2,378 875 7,646 321 542 12 OTA 1,236 12,700 694 11,345 365 603 13 OTA 865 5,640 549 5,578 532 685 14 OTA 1,130 6,389 253 3,543 130 603 15 OFA 456 8,653 159 3,211 147 365 16 OFA 442 7,833 377 6,489 230 521 17 OFC 583 6,421 590 7,345 184 475 18 OFA 345 10,054 1,050 8,897 163 295 19 OFA 321 3,890 858 6,789 208 265 20 OFA 238 3,420 665 5,780 201 385 21 OFA 643 7,804 947 8,085 220 430 22 OFA 436 4,042 635 8,988 251 541 #, Copy number/ -ACTIN copy number; OFA, oxyphilic follicular adenoma; OFC, oxyphilic follicular carcinoma; OTA, oxyphilic follicular adenoma (trabecular pattern); C, control surrounding tissue; O, oxyphilic thyroid tumor. In our study of seven fresh oxyphilic tumors, the increased Acknowledgments expression of UCP2 was probably responsible for the cou- We are grateful to C. Savagner for the statistical analysis and to K. pling defect reflected by a significant decrease in ATP syn- Malkani for critical reading of the manuscript. We thank Anne, Domin- thesis. Mitochondrial proliferation could therefore be an ique, and Florence for continuous support during the study. adaptive response to a primary nuclear abnormality, namely Received January 26, 2001. Accepted June 5, 2001. the overexpression of UCP2. However, the overexpression of Address all correspondence and requests for reprints to: F. Sav- UCP2 might itself be a response to the proliferation of mi- agner, Inserm EMI-U 00-18, Laboratoire de Biochimie et Biologie tochondria compensating for the decreased mitochondrial Moleculaire, Chu, 4 rue Larrey, F-49033 Angers cedex 01, France. ´ ATP synthesis. In the latest case, the proliferation of mito- E-mail: This work was supported by grants from l’Association pour la Re- chondria leads to overproduction of reactive oxygen species, cherche sur le Cancer. which could be counteracted by an increase in UCP2 expres- sion (26). References Another line of reasoning suggests that the metabolism in 1. Hedinger C, Williams ED, Sobin LH 1989 The WHO histological classification oxyphilic tumors has switched to a glycolytic status (12). The of thyroid tumors: a commentary on the ed. 2. Cancer 63:908 –911 decrease in mitochondrial ATP synthesis we noticed could 2. Katoh R, Harach HR, Williams ED 1998 Solitary, multiple, and familial oxy- phil tumours of the thyroid gland. J Pathol 186:292–299 lead to a shift toward anaerobic metabolism. Because the 3. Ricquier D, Bouillaud F 2000 The uncoupling protein homologues: UCP1, defect is measured in oxyphilic adenoma as well as in car- UCP2, UCP3, StUCP and AtUCP. Biochem J 345:161–179 4. Tallini G 1998 Oncocytic tumours. Virchows Arch 433:5–12 cinoma, we suggest that the metabolism switch is an early 5. Bronner MP, Clevenger CV, Edmonds PR, Lowell DM, McFarland MM, event in the oxyphilic thyroid tumor progression. Thus, the LiVolsi VA 1988 Flow cytometric analysis of DNA content in Hurthle cell ¨ adenomas and carcinomas of the thyroid. Am J Clin Pathol 89:764 –769 oxyphilic cell might be early resistant to hypoxia, which 6. Bouillaud F, Ricquier D, Thibault J, Weissenbach J 1985 Molecular approach could explain the aggressive clinical behavior of these tumors to thermogenesis in brown adipose tissue: cDNA cloning of the mitochondrial (22). uncoupling protein. Proc Natl Acad Sci USA 82:445– 448 7. DiMauro S, Bonilla E, Zeviani M, Nakagawa M, DeVivo DC 1985 Mito- In conclusion, the defective ATP synthesis we observed in chondrial myopathies. Ann Neurol 17:521–538 seven oxyphilic thyroid tumors might explain the mitochon- 8. Shoffner JM, Wallace DC 1990 Oxidative phosphorylation diseases. Disorders of two genomes. Adv Hum Genet 19:267–330 drial proliferation found in the tumor cells. Because the ex- 9. Heddi A, Lestienne P, Wallace DC, Stepien G 1993 Mitochondrial DNA pression of UCP2 was higher in all the 22 oxyphilic thyroid expression in mitochondrial myopathies and coordinated expression of nu- clear genes involved in ATP production. J Biol Chem 268:12156 –12163 tumors, compared with controls, we suggest that the oxida- 10. Flier J, Lowell B 1997 Obesity research springs a proton leak. Nat Genet tive phosphorylation coupling defect we detected may be 15:223–224 associated with mitochondrial proliferation in oxyphilic thy- 11. Ebner D, Rodel G, Pavenstaedt I, Haferkamp O 1991 Functional and mo- lecular analysis of mitochondria in thyroid oxyphil tumour. Virchows Arch B roid tumors. It would therefore be of interest to further in- Cell Pathol Incl Mol Pathol 60:139 –144 vestigate the factors involved in the transcription and rep- 12. Heddi A, Faure-Vigny H, Wallace DC, Stepien G 1996 Coordinate expression of nuclear and mitochondrial genes involved in energy production in carci- lication of mitochondrial DNA in oxyphilic tumors of the noma and oxyphil tumour. Biochim Biophys Acta 1316:203–209 thyroid gland. 13. Chretien D, Bourgeron T, Rotig A, Munnich A, Rustin P 1990 The measure-
  7. 7. Savagner et al. • ATP Synthesis in Oxyphilic Thyroid Tumors J Clin Endocrinol Metab, October 2001, 86(10):4920 – 4925 4925 ment of the rotenone-sensitive NADH cytochrome c reductase activity in 20. Cortopassi G, Shibata D, Soong N, Arnheim N 1992 A pattern of accumu- mitochondria isolated from minute amount of human skeletal muscle. Bio- lation of a somatic deletion of mitochondrial DNA in aging human tissues. Proc chem Biophys Res Commun 173:26 –33 Natl Acad Sci USA 89:7370 –7374 14. Lemasters JJ, Hackenbrock CR 1976 Continuous measurement and rapid 21. Tallini G, Ladanyi M, Rosai J, Jhanwar SC 1994 Analysis of nuclear and kinetics of ATP synthesis in rat liver mitochondria, mitoplasts and inner mitochondrial DNA alterations in thyroid and renal oncocytic tumors. Cyto- membrane vesicles determined by firefly-luciferase luminescence. Eur J Bio- genet Cell Genet 66:253–259 chem 67:1–10 22. Maximo V, Sobrinho-Simoes M 2000 Hurthle cell tumours of the thyroid. A ¨ 15. Tallini G, Carcangiu ML, Rosai J 1992 Oncocytic neoplasms of the thyroid review with emphasis on mitochondrial abnormalities with clinical relevance. gland. Acta Pathol Jpn 42:305–315 Virchows Arch 437:107–115 16. Reynier P, Chretien MF, Savagner F, et al. 1998 Long PCR analysis of human 23. Savagner F, Chevrollier A, Loiseau D, et al. 2001 Mitochondrial activity in gamete mtDNA suggests defective mitochondrial maintenance in spermato- XTC.UC1 cells derived from thyroid oncocytoma. Thyroid 11:327–333 zoa and supports the bottleneck theory for oocytes. Biochem Biophys Res Commun 254:373–377 24. Lamy FM, Rodesch FR, Dumont JE 1967 Action of thyrotropin on thyroid 17. Heddi A, Stepien G, Benke PJ, Wallace DC 1999 Coordinate induction of energetic metabolism. VI. Regulation of mitochondrial respiration. Exp Cell energy gene expression in tissues of mitochondrial disease patients. J Biol Res 46:518 –532 Chem 274:22968 –22976 25. Ricquier D, Bouillaud F 2000 Mitochondrial uncoupling proteins: from mi- 18. Welter C, Kovacs G, Seitz G, Blin N 1989 Alteration of mitochondrial DNA tochondria to the regulation of energy balance. J Physiol 529:3–10 in human oxyphil tumours. Genes Chromosomes Cancer 1:79 – 82 26. Arsenijevic D, Onuma H, Pecqueur C, et al. 2000 Disruption of the uncoupling 19. Wallace DC 1992 Diseases of the mitochondrial DNA. Annu Rev Biochem protein-2 gene in mice reveals a role in immunity and oxygen species pro- 61:1175–1212 duction. Nat Genet 26:435– 439