Circular Oligomers in Mitochondrial DNAofHumanandBeef ... · 848 Biochemistry: Paoletti et al. DISCUSSION ment indicates Thyroid mitochondria display all the enzymatic activities

Proc. Nat. Acad. Sci. USAVol. 69, No. 4, pp. 847-850, April 1972

Circular Oligomers in Mitochondrial DNA of Human and BeefNonmalignant Thyroid Glands

(nonmalignant/dimers/electron microscopy)

CLAUDE PAOLETTI, GUY RIOU, AND JACQUES PAIRAULT

Laboratory of Molecular Pharmacology and Laboratory of Biochemistry and Enzymology, Institut Gustave Roussy, 94, Villejuif, France

Communicated by Andre Lwoff, January 24, 1972

ABSTRACT Circular dimers and higher oligomers ofmitochondrial DNA have previously only been described intumor cells. This work demonstrates that such oligomersare also consistently found in a relatively high content(10-37% in weight) in nonmalignant human and beefthyroid glands. Therefore, these forms cannot be assumedto be specific for malignancy, even if they could be relatedto it in some instances.

In 1967, Clayton and Vinograd (1) made the important ob-servation that the mitochondrial DNA (mt-DNA) of humanleukemic leukocytes contained double-length (dimers) andmultiple-length (oligomers) closed structures, as well as thecatenated forms present in all tissues. This observation wasextended to some other human and experimental malig-nancies (2-6). However, the presence of these structures is notthe rule in tumor cells (reviewed in ref. 7). The occurrenceof circular dimers and oligomers has not previously been re-ported in mitochondria of nonmalignant cells (8), althoughthey have been observed in other eukaryotes (yeast and tryp-anosomes), bacteria (episomes), animal viruses, and bac-teriophages (reviewed in ref. 7). In this work, we show thatthe mt-DNA of nonmalignant thyroid cells from humans andbeef contain a high proportion of circular dimers and oli-gomers.

MATERIAL AND METHODS

The thyroids were either frozen and kept at -70° or im-mediately processed after their removal. The clinical descrip-tions of the patients are summarized in Table 1. The I'lIstorage capacity of each human thyroid was normal, exceptfor the adenoma of case 1. The samples were all serially ex-amined after histological staining, and no malignant cells wereobserved. The thyroid of case 5 and the thyroids of the beefwere histologically normal.Mitochondria were isolated and mt-DNA was extracted as

described (9). In most cases, closed-circular molecules ofmt-DNA were obtained from the lower band of a CsCl-ethidium bromide gradient and spread on a 0.1 M ammoniumacetate hypophase for electron microscopy. In some instances,mt-DNA was directly extruded from mitochondria lysed indistilled water and collected on grids after the addition of cy-tochrome c (100 ug/ml). The specimens were examined with aPhilips EM 300. The buoyant density experiments were per-

formed according to the procedure described by Vinograd andHearst (10).

RESULTS

Fig. 1 shows some typical electron micrographs of mt-DNAmolecules isolated from a human thyroid. Fig. 2 and Table 1give the length distribution (histogram) and the frequencyof each length class. mt-DNA from nonmalignant thyroids ofhumans, as well as from normal thyroids of beef, yields a highproportion of circular dimers; the frequency ranges from10 to 37% by weight of the total extracted mt-DNA.Two malignant human thyroids were also studied. The

first one was actually invaded by a metastasis from a primarylung cancer. It did not yield any circular oligomers. The secondone displayed the histological characteristics of a folliculardifferentiated epithelioma and contained 22.1% by weightof circular dimers.We found an increased frequency of circular dimers and

higher oligomers in osmotically shocked mitochondria, ascompared with purified mt-DNA. For instance, in one ex-periment not reported in Table 1, we scored 23% of circulardimers after osmotic shock, and only 16% in purified mt-DNA.Therefore, the values given in Table 1 are minimal ones;there are probably more circular dimers in vivo than thoseafter extraction. The possible cause of such variations isdiscussed elsewhere (7).The largest circular molecule measured was 24 um long (os-

motic shock). Different types of branched circular moleculeswere seen. Fig. 1 (micrograph 5) shows a double-forked struc-ture catenated in a trimer, found in human thyroid mt-DNAextruded from osmotically shocked mitochondria. The size ofeach part of this structure strongly suggests that it is a replica-tive form of a monomer according to Cairns' model. Such struc-tures were very rare. To our knowledge, it is the first report ona Cairns's replicative form included in a catenated oligomer.Cairn's structures have also been described in mt-DNA byKirschner et al. (11). We have very seldom recorded forkedcircular structures with free branches.

Small loops on circular monomers and dimers were frequent.They are very probably identical to the displacement loops(D-loops) recently described by Kasamatsu et al. in mt-DNAof mouse cell lines (12) and by Arnberg et al. (13) in mtDNAof chicken liver. Our data will be reported in detail elsewhere.The buoyant densities of human and beef mt-DNA were,

respectively, p = 1.706 g/cm3 and p = 1.701 g/cm3 (Fig. 3).

847

Abbreviation: mt-DNA, mitochondrial DNA.

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848 Biochemistry: Paoletti et al.

DISCUSSION ment indicates

Thyroid mitochondria display all the enzymatic activities the formation ofcharacteristic of these organelles isolated from other mam- are known to mmalian tissues, and their respiratory chain phosphorylation tion of thyroxinsystem is equally responsive to the standard electron- and over of mt-DNIenergy-transfer inhibitors (14). The presence of circular oligo- mt-DNA in themers in DNA is their only known unusual feature. No argu- half the rate of ti

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that thyroid hormones could be involved incircular oligomers. However, these hormonesodify the metabolism of mt-DNA. The injec-into rats induces a rapid increase of the turn-& in their livers. In contrast, the turnover ofliver of thyroidectomized animals occurs athat in the liver of normal rats (15).

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FIG. 1. Electron micrographs of mt-DNA from human thyroid. 1-4: Chemically extracted mt-DNA (lower band of ethidium bromide-OsCi gradient). 1: Circular dimer. 2: Twisted monomer and catenated tetramer, made of two circular dimeric subunits. 3: Circular trimer.4: Catenated hexamer (three interlocked circular dimeric subunits). 5: DNA extruded from osmotically shocked mitochondria. Replicativeform of the monomeric part of a catenated tetramer. The arrows indicate two branching points on the same structure. The distances be-tween them are 1.65 Mm, 3.35 pim, and 3.75 pum.

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Proc. Nat. Acad. Sci. USA 69 (1972)

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Thyroid Mitochondrial DNA 849

TABLE 1. Relative frequency of monomers and oligomers in purified mt-DNA of nonmalignant human and beef thyroids

mt-DNA (% by weight)

Exp. molecules Dimers HigherOrigin no. scored Monomers Catenated Circular oligomers

Follicular adenoma 1 687 47.5 13.0 25.0 14.5Microfollicular* polyadenoma 2 308 32.3 18.7 37.0 12.0Thyroiditis 3 451 73.6 6.4 11.7 8.3Basedow (Hyperthyroidism)t 4 173 40.4 9.9 32.0 17.7"Normal" humant 5 820 55.8 12.5 25.2 6.5Normal beef 6 220 70.0 7.7 17.8 4.5Normal beef 7 293 84.1 5.5 10.1 0.3

* Exp. 2 was performed with the tissue surrounding the adenoma nodula used in expt. 1. This tissue was apparently healthy and able tostore 13II; however, histologic examination revealed several very small adenomatous foci.

t This patient received an antithyroid drug (Neomercazole) months before exeresis.t This patient was treated with corticosteroids, vincristine, and adriamycin for an acute lymphoblastic leukemia; when he died, the

leukocyte count was 600 per mm3.

The main conclusion of this work is that circular dimers andoligomers occur in the mt-DNA of nonmalignant tissues.Until now, they were thought to be characteristic of malig-nancy, and had not been found in animal cells other than incancer.To complete this conclusion, it should be stressed that the

existence of such oligomers in cancer cells is not a general rule.They have neither been found in some tumors of human origin[nephroblastoma and neuroblastoma (6), metastatic pul-

monary epithelioma (this work), Burkitt lymphoma (5), andHeLa cells (16) 1, nor in some experimental tumors (reviewedin ref. 7). However, no significance other than indicative couldbe ascribed to such negative correlation, since the possibilityalways exists of a loss of mt-DNA during extraction. More-over, the content of circular oligomers of mt-DNA in mousefibroblasts (L cells) depends strongly on the metabolic stateand the environmental conditions of the cells (3, 5).Once more, a search for a general and specific relationship

BEEF THYROID

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0.8 1.0 1.2 1.8 2.0 2.2 2.8 3.0 3.2 0.8 1.0 1.2 1.8 2 2.2 2.8 3.0 3.2

RELATIVE LENGTH

FIG. 2. Normalized contour lengths of free circular DNA and subunits of catenated oligomers in mt-DNA of thyroids. Human: (a)Exp. 1; b) Exp. 4; c) Exp. 3; d) Exp. 5. Beef: Exp. 6. (see Table 1). The abscissas were normalized to the mean length of the monomeric,dimeric, and trimeric populations. N is the number of molecules measured.

0.8 1.0 1.2 1.8 2.0 2 2.2 2.8 3.0

Proc. Nat. Acad. Sci. USA 69 (1972)

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850 Biochemistry: Paoletti et al.

1.701 1.706 1.742

a

b

FIG. 3. Buoyant density of mt-DNA from human and beefthyroids in CsCl gradient. (a) Human mt-DNA of thyroid poly-adenoma (expt. 2 in Table 1). (b) Beef mt-DNA. The runs were

performed in the Spinco model E analytical ultracentrifuge for 20hr at 250 and 44,770 rpm. The UV photographs were scanned witha Joyce-Loebel microdensitometer. The buoyant density of mt-DNA is expressed relative to the density of Bacillus subtilis phage2c DNA, taken as 1.742 g/cm3; a value of 1.710 g/cm3 for E. coliDNA was used as a reference.

between the cancer process and some biochemical distur-bance(s) leads to a disappointing result, even if the presence

of circular dimers in mt-DNA from leukocytes of humanleukemic leukemia (1, 6) still deserves much attention.

We thank Dr. J. B. Le Pecq and Dr. Y. Lanni for criticalreading of the manuscript and Drs. R. Gerard-Marchant and C.Micheau for histological work. The excellent technical assistanceof Miss M. Gabillot is acknowledged. Our group is part of theLaboratoire de Pharmacologie Moleculaire associ6 au CNRS.This work was supported by "INSERM" and the "LigueNationale Frangaise contre le Cancer."

1. Clayton, D. A. & Vinograd, J. (1967) Nature 216, 652-657.2. Clayton, D. A. & Vinograd, J. (1969) Proc. Nat. Acad. Sci.

USA 62, 1077-1084.3. Nass, M. M. K. (1969) Nature 223, 1124-1129.4. Nass, M. M. K. (1969) J. Mol. Biol. 42, 521-528.5. Nass, M. M. K. (1970) Proc. Nat. Acad. Sci. USA 67,

1926-1933.6. Paoletti, C. & Riou, G. (1970) Bull. Cancer 57, 301-334.7. Paoletti, C. & Riou, G., "Mitochondrial DNA in malignant

cells," Progress in Molecular and Subcellular Biology, ed.Hahn, F. E. (Springer Verlag Publ.), Vol. 3, in press.

8. Clayton, D. A., Smith, C. A., Jordan, J. M., Teplitz., M. &Vinograd, J. (1968) Nature 220, 976-979; Piko, L., Blair,D. G., Tyler., A. & Vinograd, J. (1968) Proc. Nat. Acad.Sci. USA 59, 838-845.

9. Riou, G. & Lacour, F. (1971) Biochimie 53, 47-49.10. Vinograd, J. & Hearst, J. E. (1962) in Fortschritte der

Chemie Organischer Naturstoffe, ed. Zechmeister, L. (SpringerVerlag, Vienna), Vol. 20, pp. 372-420.

11. Kirschner, R. H., Wolstenholme, D. R. & Gross, N. J.(1968) Proc. Nat. Acad. Sci. USA 60, 1466-1472.

12. Kasmatsu, H., Robberson, D. L. & Vinograd, J. (1971)Proc. Nat. Acad. Sci. USA 68, 2252-2257.

13. Arnberg, A., Van Bruggen, E. F. J., Ter Schegget, J. &Borst, P. (1971) Biochim. Biophys. Acta 246, 353-357.

14. Tyler, D. D. & Gonze, J. (1967) in Methods in Enzymology,eds. Estabrook, R. W. & Pullman, M. E. (Academic PressPubl.), Vol. 10, pp. 101-103.

15. Gross, N. J. (1971) J. Cell Biol. 48, 29-40.16. Radloff, R. W., Bauer, W. & Vinograd, J. (1967) Proc.

Nat. Acad. Sci. USA 57, 1514-1521; Hudson, B. & Vinograd,J. (1967) Nature 216, 647-652.

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