Mitochondrial Inclusions in Human Cancer of the Gastrointestinal Tract

[CANCER RESEARCH 37, i556-i563, May 1977) Mitochondrial Inclusions in Human Cancer of the Gastrointestinal Tract

Vittorio Marinozzi,2 Massimo Derenzini,3 Francesco Nardi, and Pietro Gallo

Laboratorio di Microscopia Elettronica, 1Â°lstitutodi Anatomia Patologica, UniversitÃ¢di Roma, Policlinico Umberto 1Â°VialeRegina Elena, 324, 1-00161Rome, Italy

SUMMARY den to discover their chemical nature. The results are me ported here, and the possible relationship between DNA Four cases of adenocancinoma of the gastrointestinal and the electron-dense inclusions is discussed with refer tract have been examined. Lange electron-dense mitochon ence to recent literature on the subject. dnial inclusions were found in the mitochondria of many cells; after detailed histochemical tests they could be classi fied into two types. The first type of inclusion consisted of MATERIALS AND METHODS clusters of electron-dense, calcium-containing granules linked to a glycoproteic substrate. These inclusions were The material examined by us was obtained from a gastric always associated with cnistae and were found in the mito carcinoma, a metastatic tumor of the liven from a gastric chondnia of cells that showed clear signs of degeneration. carcinoma, a carcinoma of the sigma-rectum, and a meta The second type of inclusion was found much more fre static tumor of the liver from an intestinal carcinoma. In quently and consisted essentially of phospholipids of which each case the material, along with fragments of normal electron density was strictly osmium dependent. Their tissue, was removed during a surgical operation. The speci structure was usually at least partly lamellan, but in some mens were fixed in 3 ways: (a) with 1% 0504 in 0.01 M cases it was homogeneous throughout. It is hypothesized phosphate buffer, pH 7.4, for 1 hr; (b) with 4% formaldehyde that inclusions of the second type may have the same bio mixed with an equal volume of 2.5% glutamaldehyde in 0.01 logical role as the morphologically identical inclusions M phosphate buffer for 2 hm; and (c) with 4% formaldehyde found in the mitochondnia of brown fatty tissue in the pen for 2 hr followed by i hr postfixation with 1% 0504, both natal rat and in yeasts during glucose repression or anaero buffered as above. biosis. The resemblance between the homogeneous variety After fixation, the specimens were dehydrated and then of inclusions within the second type and the mitochondnial embedded in an epon-amaldite mixture. Ultrathin sections inclusions recently described in human leukemic lympho from undecalcified specimens were examined under the blasts and monoblasts has been stressed to bring out the electron microscope: (a) without treatment of any kind, (b) need for a histochemical check on the supposedly viral after â€œstainingâ€•withumanylacetateand leadcitrate, (C)after nature of the latter inclusions. decalcification by 15-mm flotation on 0.1 N HCI or 0.2 M EDTA, pH 7, and (d) after decalcification as in c and posts taming with unanyl and lead or with 1% phosphotungstic INTRODUCTION acid in 0.1 N hydrochloric acid (35). Some sections from osmium-fixedspecimenswere alsoexamined afterbeing Of all cytoplasmic organelles, the mitochondnia in neo treated with 2% H2O2for 20 mm; this removes the reduced plastic cells display the severest damage ultrastructurally osmium linked to the organic structures (40), thus allowing (5), biochemically (61), and metabolically (45). This has comparison between osmium- and aldehyde-fixed struc aroused growing interest in the role of chondniome in tunes. To avoid acid-metal interaction and contamination of oncogenesis (4), an interest strengthened by reports of the ultrathin sections on the grids, both decalcification with virus-like inclusions in the mitochondnia of human leukemic HCI and phosphotungstic acid staining were carried out on lymphoblasts and monoblasts (51-54, 59). free-floatingsections(33,34). We have carried out an ultrastructumal study of 4 cases of carcinoma of the human gastrointestinal tract. The 2 main findings were: (a) the presence of large electron-dense in RESULTS clusions and (b) the increased prominence of DNA as corn pared with controls. A histochemical study was performed There were no appreciable differences among the mito on the ultrastructume of the mitochondnial inclusions in or chondnia in the 4 tumors examined. The mitochondnia ap peared in a great variety of shapes and sizes. Some were

I This work was supported by grants from the Italian National Research swollen, and the matrix of these was clearer than in con Council (CNR). trols. Others had matrices of normal electron density, but 2 To whom requests for reprints should be addressed. their cmistae were badly damaged. Some cristae, for in 3 Present address: Istituto di Patologia Generale dell'UniversitÃ di Be logna, Bologna, Italy. stance, appeared to be swollen and shorter than normal; Received May 10, 1976; accepted November 8, 1976. more rarely, they were tubular. The mitochondnial matrix

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Downloaded from cancerres.aacrjournals.org on October 6, 2021. © 1977 American Association for Cancer Research. Mitochondrial Inclusions in Human Cancer Cells contained large, electron-dense bodies which could be di controls from normal mucous membrane. DNA was easily vided into 2 types on the basis of morphological and, espe observable in the form of rod-like structures in sections cially, histochemical grounds. stained with umanylacetate and lead citrate, regardless of The 1st type of inclusion consisted of bodies that were the type of fixation used. The structures were rather large spheroid , with diameters between 600 and 1400 A. These (up to 500-A thick), and thinner filaments branched out inclusions were found only in swollen mitochondnia with from their ends. These structures were found at the center matrices of low electron density. They were usually found of the areas in which the mitochondnial matrix was clearest near mitochondnial cnistae (Fig. iA). At high magnification, (Figs. 3C and 4, A and B). DNA filaments were often found the inclusions of this type appeared as clusters of very in mitochondria that contained osmiophilic inclusions, in electron-dense granules often enclosing a less electron which case the inclusions were almost always found near dense area in which only a few granules could be found the clear DNA-containing areas (Figs. 3C and 4, A and B). (Fig. iB). The fact that these bodies consisted of clusters of In sections taken from material fixed in aldehydes only electron-dense granules was particularly evident in speci and stained with lead at high pH or with uranyl acetate and mens that had been fixed in aldehydes only and had not lead citrate, many electron-dense granules, about 150 A in been stained (Fig. iC). The mitochondnia that contained diameter, could be observed (Fig. 40). They were identifia these inclusions were generally found in cells degenerating ble as mitonibosomes. There were many more mitonibo near areas of necrosis and in cells with partially interrupted somes in the cancer cells than in the controls. plasma membranes (Fig. 1A). The histochemical character istics of this type of inclusion were: (a) The mitochondnia contained inclusions of high electron density, irrespective DISCUSSION of the fixation method used (Fig. 1, A and C); (b) in sections treated with EDTA or 0.1 N HCI the inclusions disappeared, The main characteristics of mitochondnia in the 4 neopla and extremely clear areas were left with diameters that were sias studied were: (a) the presence in the matrix of electron the same as those of the inclusions (Fig. 1D); (c) after dense inclusions different from normal dense mitochondnial sections were treated as in b and then stained with uranyl bodies, and (b) a large increase in the amounts of nucleic acetate and lead, the inclusions reappeared with character acids present. Mitochondnial inclusions were shown histo istics that were almost identical to those of untreated sec chemically to be of 2 types. The 1st type was intrinsically tions; and (d) after sections had been treated as in b and electron dense and disappeared when sections were treated stained with phosphotungstic acid, round clusters of elec with 0.1 N HCI or EDTA. When such treatment was followed tron-dense material resembling those in untreated mito by staining with phosphotungstic acid or umanylacetate and chondmia were found. Treatment of sections with H202 had lead citrate, the inclusions reappeared, displaying the same no effect on the electron density of the inclusions. characteristics as in nontreated sections. Similar intninsi The 2nd type of inclusion consisted of dense bodies of cally electron-dense mitochondnial inclusions were me highly variable structure, with diameters ranging between ported under a variety of physiological conditions (12, 23, 600 and 2000 A. In most cases, they had a composite struc 38,39)and pathologicalstates(8,9,14-17)and inisolated tume comprising a number of lamellar shapes which were mitochondnia incubated in a calcium-containing solution sometimes arranged in irregular whorls (Fig. 2,4)and some (13, 20, 21, 44). The morphological and histochemical data times compressed in apparently parallel layers (Fig. 2B). In have generally been considered convincing evidence that some cases, part of the inclusion, quite often its central the inclusions consist of calcium salts which are probably zone, appeared to be completely homogeneous (Fig. 2B). linked with a glycoproteic substrate (7). Constant associa More rarely, inclusions appeared as spheroid bodies of tion with cmistae and a granular appearance are 2 other homogeneous density and almost unvarying size (Fig. 2C). typical features of this type of inclusion. In the casesinvesti The histochemical characteristics of the 2nd type of inclu gated by us, these calcium-containing inclusions were con sion were: (a) electron density was strictly dependent on stantly found in damaged mitochondnia in degenerative osmiophilia, and the inclusions were no longer observable cells. This is in agreement with reports by other authors of after fixation in aldehydes only; (b) treatment of osmium damaged mitochondnia in cells at various stages of degen fixed sections with H2O2led to the disappearance of the emation (9, 19, 24, 47, 48). In the present investigation also, inclusions, leaving extremely clear areas with diameters intramitochondnial calcification may be considered a sign equal to those of the inclusions (Fig. 3A); and (c) treatment of cellular damage. of sections with EDTA or 0.i N HCI produced no appreciable Inclusions of the 2nd type differed from those of the 1st change in their electron density (Fig. 3B). This type of type in morphology and histochemical properties. The elec inclusion was found in mitochondmia containing few cnistae, tron density of these bodies was not intrinsic but was wholly independently of whether the mitochondnial matrix was dependent on their osmiophilia. They were not observable swollen. These inclusions were often, but not always, found in sections fixed in aldehydes only, and they disappeared in near the inner membrane of mitochondnia. They were never sections taken from fragments fixed in 0504 when these found together with the ordinary dense mitochondnial bod were treated with H2O2.This characteristic is also found in ies and, with mareexceptions, were not found near inclu normal dense mitochondnial bodies, and it is probable that, sionsofthe1sttype. like the latter, these inclusions are made up of organic Increased prominence of DNA and a large increase in the compounds, most likely unsaturated lipids (1, 37, 49). number of mitoribosomes were found in mitochondnia and intracytoplasmic whorls of the type shown in Fig. 2C were in all 4 types of tumors when a comparison was made with occasionally found. This might suggest that the osmiophilic

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Downloaded from cancerres.aacrjournals.org on October 6, 2021. © 1977 American Association for Cancer Research. V. Marinozzi et a!. lamellar inclusions observed by us in the mitochondnia of mitochondnia in which levels of DNA and numbers of mitomi tumor cells could, like those studied by Cumgy (18) in the bosomeswere exceptionally high. The high content of mito hepatocytes of chick embryos, be the outcome of the mo chondmial DNA and RNA in neoplastic cells has been dem lecular rearrangement of phospholipids drawn out from onstrated morphologically and biochemically by a number preexisting membranes during aldehyde fixation, with later ofauthors(6,10,28,36,43,62)and has been attributedto fixation and staining by 0504. It seems, however, that this the intense mitochondmiogenesis that occurs in tumor cells hypothesis can be excluded because: (a) the osmiophilic because of the rapid division of cells. The common associa inclusions found in tumor cells had a more variable and tion of mitochondnial DNA with lamellar osmiophilic inclu complex structure and a much higher electron density than sions again points to the analogy between neoplastic cells the membranous whorls induced by aldehyde fixation; (b) and yeast during glucose repression or anaemobiosis. In they were sometimes found near mitochondnial cnistae, but these 2 cases, mitochondnia contain both lamellar osmio never touching them or touching the outer mitochondnial philic inclusions and a large amount of DNA. Both Yotsuy membrane; and (c) their presence could not be due to anagi (63) and Swift and Wolstenholme (58) have stressed aldehyde fixation because they were also found in tissues the constant association between mitochondmial DNA and fixed in 0504 alone. Large osmiophilic mitochondnial bod lamellamosmiophilic inclusions, as well as their close topo ies have been found in a large variety of organisms (2) and graphicalrelationship. in neoplastic cells (25, 27, 29, 46). A lamellamarrangement In the material studied by us, the association between the has also been reported by other investigators (11, 29, 41, DNA and osmiophilic inclusions was common but not invar 42). iable, a finding which leads us to believe that their coexist The inclusions found in the brown fatty tissue of the ence in a single mitochondrion may be purely coincidental. perinatal rat offer a useful model for the study of the biogen The homogeneously dense, osmiophilic mitochondnial in esis and role of this 2nd type of inclusion. In such tissue, clusions reported here deserve further attention because of osmiophilic intramitochondnial inclusions appear during a their resemblance to what have recently been reported as period of intense mitochondmiogenesischaracterized by a â€œvirus-likeâ€•intramitochondnialinclusions in human leu low level of enzymatic activity and a maximum concentra kemic monoblasts and lymphoblasts (51-54, 59). This find tion of phospholipids. As mitochondniogenesis becomes ing, together with the morphological demonstration of in less intense, amounts of electron transport enzymes in tramitochondnial viruses in cells infected with oncogenic crease, and the osmiophilic bodies gradually disappear. It viruses (22, 26, 29-32), led to the suggestion that intra seems that they are replaced by the normal dense bodies in mitochondnial virus infection may play a decisive mole in the matrix, and the number of cnistae in each mitochondnial oncogenesis (26, 50). However, it must be noted that the profile rises. These findings suggest that inclusions of this supposed virus particles found in leukemic cells have not type are the outcome of a lack of synchrony between the yet been studied histochemically. Thus the possibility that synthesis on incorporation of some electron transport en they too may consist of phospholipids cannot be excluded. zymes in the membrane and the production of other mem Further research is being carried out in our laboratory to brane components. According to this interpretation, insuffi determine the relative frequencies of osmiophiiic intramito ciently high concentrations of electron transport enzymes chondnial inclusions in neoplasias affecting a wide variety lead to the accumulation of other membrane components in of different organs and tissues. the form of large osmiophilic inclusions. When enough electron transport enzymes become available, the material ACKNOWLEDGMENTS stored in the granules contributes to the formation of the membrane (3, 55, 56). There is a strong resemblance be The authors wish to thank Dr. A. L. Johnson of Pisa University for his help tween the intramitochondnial inclusions found in the brown with translation. fatty tissue of the peninatal matand the inclusions reported here. This suggests the hypothesis that the latter, like the REFERENCES former, may be formed in concomitance with a metabolic shortfall of electron transport enzymes with respect to the 1. AndrÃ©,J., and Marinozzi, V. Presence dans les Mitochondries, de Parti cules Ressemblant aux Ribosomes, J. Microscop., 4: 615-626, i965. available supply of phospholipoprotein. It is, in fact, well 2. Barnard, T., and Afzelius, B. A. The Matrix Granules of Mitochondria: a known that a low respiration mateand an unusually intense Review. 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Fig. 1. All the micrographs in this figure are from the same adenocarcinoma of the sigma. Almost all the mitochondria in the cancer cells contain bodies, the volumes of which are much higher than those of normal dense bodies in the mitochondrial matrix. In A, it is shown clearly that inclusions are always located near cristae. Arrows, zone of lysis of plasma membrane. x 20,000. In B, the electron-dense bodies are evidently made up of clusters of granules which often form a ring around a clear center. x 13,000. The characteristic granular appearance of the electron-dense material is seen more distinctly in unstained sections from the specimens fixed in aldehydes only, as in C. x iio,500. In D, the extremely clear areas in the mitochondria correspond to the site of dense bodies extracted by treatment of an ultrathin section with 0.1 N HCI. x 32,500.

1560 CANCER RESEARCH VOL. 37

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@ . @ :. lb â€˜@ Fig. 2. All the mlcrographs in this figure are from a metastatic tumor of the liver from a gastric adenocarcinoma. The specimens were fixed In formol and glutaraldehyde and postfixed in osmium . The ultrathin sections were stained with uranyl acetate and lead . In A, large electron-dense inclusions with a lamellar structure are seen In the matrices of 2 mitochondria. Arrow, a DNA fiber. x 97,500. In B, 2 inclusions are seen in the same mitochondrion. Their structure is mainly lamellar, but in places the electron-dense material is homogeneous. x i6@,000.All the mitochondria seen in C contain many electron-dense bodies of almost equal size and of almost homogeneous electron density. Arrow, a DNA fiber. An extramitochondrial membranous whorl (W) is visible. )<65,000.

MAY 1977 i56i

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Fig. 3. All the micrographs in this figure are from a metastatic tumor of the liver from a gastric adenocarcinoma. In A, treatment of ultrathin sections with H202,followed by staining with uranyl acetate and lead, leads to the disappearance of the mitochondrial inclusions. Extremely clear areas appear in their place. x 65,000. In B, treatment of the sections with 0.1 N HCI shows no appreciable change in the electron density of the mitochondrial inclusions. This micrograph also shows that a single cell can contain large mitochondrial inclusions with homogeneous electron density and others with a partly lamellar appearance (arrows). x 65,000. In C, part of a neoplastic cell shows mitochondria containing lamellar osmiophilic bodies and DNA. x 70,000.

1562 CANCER RESEARCHVOL. 37

MitoChondrial Inclusions in Human Cancer Cells

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Fig. 4. A and B are from a gastric adenocarcinoma. C is from an adenocarcinoma of the sigma. Where osmiophilic mitochondrial bodies and DNA fibers are found in the same mitochondrion (A and B), the osmiophilic bodies always lie at the periphery of the clear zone containing DNA fibers. A, x 100,000; B, x 100,000. In C, the very high number of mitoribosomes in the mitochondria of cancer cells is seen clearly in sections from specimens fixed in aldehydes only and stained only with lead citrate. x 105,000.

MAY 1977 1563

Vittorio Marinozzi, Massimo Derenzini, Francesco Nardi, et al.

Cancer Res 1977;37:1556-1563.

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