Comparative Study of the Cytoplasmic Organelles of Epithelial Cell Lines Derived from Human Carcinomas and Nonmalignant Tissues

[CANCER RESEARCH 40, 803-81 7, March 19801 0008-5472/80/0040-0000$02.00 Comparative Study of the Cytoplasmic Organelles of Epithelial Cell Lines Derived from Human Carcinomas and Nonmalignant Tissues

E. Louise Springer

Donner Laboratory, University of California, Berkeley, California 94 720

ABSTRACT tissues, tissue peripheral to carcinomas, and carcinomas has been described in recent publications (44, 55). Briefly, 3 mor The cytoplasmic organeblesof 16 human epithebialcell lines phobogical classes of cells were described, normal, abnormal, have been characterized by electron microscopy. The cell lines and very abnormal. The normal cells were nearby uniform in were derived from normal, nonmalignant tissues of cancerous size, shape, and staining properties; the cells from carcinomas organs and from primary and metastatic carcinomas. Every cell were altered and highly variable in size, shape, and staining section on a grid which contained a cleanly defined nucleus, properties; while those from tissue peripheral to carcinomas nucleolus, and cytoplasm was scored blindly utilizing a check were intermediate for these characteristics. Although the mom list of markers. Mitochondniab pleomorphism was expressed phobogical changes were consistently more aberrant in the slightly by normal, to variable degrees by lines derived from malignant cell lines, they were difficult to quantitate. At the nonmalignant tissues of cancerous organs, and to a much ultrastructunal level, alterations of the nucleus and mitochon greater extent by all lines derived from malignant tissues. dna were noted in all of the tumor-derived lines (55). However, Hypertrophied mitochondmiaand longitudinal cnistabarrange there were subtle or overlapping morphological characteristics ment were found in almost all the malignant lines, but not in among the tumor cell lines that precluded quantitative evalua any lines derived from nonmalignant tissues of cancerous tion. Therefore, a more systematic and quantitative ultrastruc organs or from normal tissues. Although malignant and non tural study of this series of human cell lines was initiated. malignant cell lines could not be distinguished using microfila Thirty-eight ultrastructunal characteristics possibly associated ment bundles (stress fibers) and microtubules as markers, this with cancer (19, 54), as well as characteristics associated with may reflect the effect of insufficient cell spread on the prepar metabolic activity were incorporated into an objective appraisal ative procedures used. All the lines appeared differentiated system for assessing the degree of expression of each char and showed slightly to moderately developed Golgi and smooth actenistic in coded specimens. The nuclear analysis of these and rough endoplasmic reticula. There were no significant human cell lines (56) showed that nuclear bodies and penichno ultrastructural differences in cells at different passage bevelson matin granules were found only in cell lines derived from subconfluent and confluent tumor cells; however, more tight carcinomas, while nuclear envelope dilation was seen in cell junctions were observed in confluent than in subconfbuent lines derived from both carcinomas and tissue peripheral to normal cells. carcinomas, but not in any normal lines. Chromatin margina tion, nuclear indentation, nucleolar fibnibbar centers, and nu INTRODUCTION cleolar manginationwere expressed slightly by the normal lines, to variable degrees by the lines derived from tissue peripheral Studies on the in vitro properties of rodent epithelial cells to carcinomas, and to a much greater extent by all carcinoma have consistently shown subtle morphological changes after derived lines. carcinogen treatment. These light microscopic observations The cytopbasmic portion of the objective appraisal system made on living and fixed-stained cultures showed several fea observations of these human cell lines is presented in this tumes:reduced intercellular cohesiveness (29, 68) and altered paper. colony formation (64, 68); pleomomphismof cell size and shape (29, 40, 41 , 65, 68), as well as nuclear size and shape (41, MATERIALS AND METHODS 52) increased nucleam:cytoplasmic ratio (40, 68); prominent, enlarged, or numerous nucleoli (41 , 52, 68); abnormal mitoses Cell Culture. The cell lines2and their tissue sources are (29, 52); and elongation of cells (68). Cytopbasmic changes listed in Table 1. All cell lines, except HeLu, were initiated at other than basophilia (52) have not been found at the light the Cell Culture Laboratory, University of California, Naval microscope level because standard fixation procedures do not Biological Laboratories, Oakland, Calif. HeLu was generously preserve cytoplasmic structures and many of the onganebbes provided by Dr. Daniel Rifkin, Rockefeller University, New York, are below the resolution limits of the microscope. Comparative N. Y. The normal epithebiabcell lines were derived from fetal studies of morphology at the light microscope bevelhave been intestine and an adult bladder from a patient with prostate difficult to quantitate. carcinoma. Two normal fibroblastic cell lines, from fetal lung Similar morphological differences at the light microscopic and foreskin were included also. Three lines derived from level have also been noted in human epitheliabcells in culture. kidney tissue peripheral to a carcinoma of the same organ were The morphology of human epithebialcells cultured from normal evaluated separately since hyperplasias are often found in the

@ I This work was supported by Contract CP-7051 0 from the National Cancer 2 Nomenclature used conforms with that of Federoff (1 4): a cell line' â€˜arises @ Institute. from a primary culture at the first subculture, while an established cell line is Received March 15, 1979; accepted November 26, 1979. one which has demonstrated the potential to be subcultured indefinitely.

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Table 1 History of patients and biopsy specimens (yr)RaceDiagnosisCarcinomas785TM58ColonDesignationSexAge

carcinoma675TMNo carcinoma761 informationColon (kidney)769TF59NTransitionalTM65C9Transitional cell carcinoma (urethra)578TF74CCarcinosarcoma cell carcinoma breast766TM64CPancreatic of carcinoma metastatic to lymph node746TM74CStomach muscle700TM61CCarcinoma carcinoma metastatic to (probably colon, intestine, or pan hip696TM430Adenocarcinoma creas) metastatic to (probably colon, intestine, or sacrumPeripheral pancreas) metastatic to

tissue of carcinoma tousorgans699KM60CNormal kidney tissue from patient with renal carcinoma761 cell KM65CNormal kidney tissue from patient with transi tional cell carcinoma kidney (same patient 761T)71 as 5KM76Normal kidney tissue from patient with renal carcinomaNonmalignant

tissues6771nt3â€”4 disorder68Olnt3â€”4 mo. fetusFamilial history of immune syndrome741nt3â€”4 mo. fetusFamilial history of Wiskott-Aldrich abnormalities767BLM45CNormal mo. fetusTherapeutic abortion, no known bladder mucosa from patient with carcinomaa prostate

c, Caucasian; N, negro; 0, oriental. uninvolved tissues of carcinomatous organs (13, 66). The so that the electron microscope operator was not aware of the malignant lines were derived from primary carcinomas of the cell source. To ensure that a cell was scored only once, the rectum, colon, breast, and transitional cells of the urethra and serial sections were surveyed to locate and identify each cell kidney, as well as metastatic lesions of pancreas, stomach, profile in the randomly chosen sample; then every cell in the and 2 lines derived from metastatic tissue of unknown origin. sample was evaluated. Scoring was accomplished by identify The growth medium used was Dubbecco's modification of ing the presence, absence, or degree of expression of various Eagle's medium (Grand Island Biological Co., Grand Island, cytoplasmic ultrastructunal characteristics for every cell section N. Y.; No. 196G) containing glucose (4.5 g/Iiten), supple showing a nucleus, nucleolus, and cytoplasm. Although most mented with 10% fetal bovine serum and insulin (10 @zg/ml; of the scoring was done directly at the electron microscope Calbiochem, San Diego, Calif.). Unless otherwise indicated, console, some photographs were taken of each specimen. cells were harvested at confluence. All of the lines, both tumor Similar results were obtained when the photomicnographs were and those derived from nonmalignant tissue, grew slowly in scored blindly. culture (44). There was no difference in growth rate as a Statistical Analysis. Tests for statistical significance were function of nuclear (56) and cytoplasmic ultrastructure. made using the Fisher exact probability test (43). A difference Electron Microscopy. The cell lines cultured in Falcon flasks was considered significant if p was 0.025 or less. were fixed in situ with 2.5% glutarabdehyde in 0.1 M sodium cacodylate buffer (pH 7.3) at room temperature for 3 hr, rinsed RESULTS with buffer, and fixed with 2% osmium tetroxide in the same buffer for 2 hr at room temperature. Both fixatives were nou Plasma Membrane Specializations. Three features of the tinely monitored for any indication of deterioration which could plasma membrane were evaluated: microvilbi, desmosomes, adversely affect cell preservation, i.e. , pH of glutanaldehyde and tight junctions. The microvibli which occur on the apical and color changes of the osmium tetnoxide. En b!oc staining and lateral surfaces of the cell as finger-like projections of the with ethanolic uranyl acetate was followed by stepwise dehy plasma membrane 1000 to 1500 A in diameter were scored. dration with ethanol and embedment in Spurn on Epon. The Microvilli located lateral between cells were often interdigitated embedded cultures were examined by light microscopy, and as shown in Fig. 1A, which also depicts a desmosome identified areas were randomly selected for sectioning without consid by the electron-dense plaques lining the inner surfaces of the eration of nuclear or cellular morphology. Ultrathin sections cut adjacent cell membranes to which tonofibnibsare attached. Fig. perpendicular to the plane of growth were poststained with 1B illustrates a tight junction which occurs when the plasma aqueous uranyl acetate and lead citrate. The serial sections membranes between 2 cells are fused by their outer leaflets so were arranged parallel to each other on copper mesh grids to that extracellulan space is obliterated (59). facilitate identification of individual cell profiles and to prevent The accumulated data on the ultrastructune of the plasma repeated analysis of the same cell. The specimens were coded membrane specializations are summarized in Chart 1. A dot

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indicates the evaluation of a single cell section for a given and 27F, have little Golgi on SER but possess the expected property. Each line was tested at least once between passages well-developed RER characteristic of this cell type. There were 4 and 20. A few lines were tested at more than one passage no differences among the epithelial on fibroblastic cell lines and, except for early passage 4 of the colon line 675T, the when they were scored for free nibosomes, empty or filled data at different passage levels were similar, including one vacuoles, or lipid droplets which were abundant in all the cell metastatic line (700T) which retained its ultrastructumal prop lines. erties for 69 passages. Microvilli were present in all of the Mitochondria. Mitochondmiaare particularly sensitive to en epithelial cell lines with the exception of the fetal intestine vironmental changes and to processing procedures for ultra (74Int). The fibmoblasts derived from fetal lung (HeLu) had structural examination (8). Standardized methods for growth microvilli; the foreskin line (27F) did not. Some but not all of and processing were used in this study in order to establish the cell lines had desmosomes or tight junctions in some consistency in any artifactuab changes. The degree of pbeo sections.Thisobservationisexpectedsinceeveryverticalcut morphism assigned mitochondria depended upon the extent of need not traverse an area with a desmosome or tight junction. outer membrane alteration, cnistal disorganization and shape Desmosomes were seen in all of the cell lines except the deviation from the standard rod configuration. Fig. 3A depicts

transitional cell carcinoma-derived line (769T), the line derived , â€˜nonpleomomphic,' , rod-shaped mitochondnia with clearly de from a metastatic tumor (696T), and the normal fetal intestine fined and closely apposed outer membranes and intact cnistae line (74lnt); while tightjunctions were absent from only one cell organized perpendicular to the long axis of the mitochondnion.

line, 699K. Both desmosomes and tight junctions were absent . â€˜Slight' â€˜pleomorphism is characterized by poorly defined outer from the 2 fibroblast lines. membranes, some intact or organized cnistae, and distended Cytocavitary System and Associated Organebles. There areas in otherwise mod-shapedmitochondnia creating â€˜â€˜bowling are few ultrastructumal markers that specifically identify the pin' â€˜orâ€˜â€˜V'â€˜forms(Fig. 3B); while â€˜â€˜moderately'â€˜pleomomphic differentiated epithelial cell in culture (16). However, using as mitochondnia displayed outer membrane buckling (arrow) on a model those epithelia cells composing the organ of origin, separation, cmistaldisorganization, and shape distortion (Fig. the degree of expression of Golgi, SEA3 and RER are reason 3C). In comparison, 2 abutting nonpleomorphic mitochondnia able guide lines for distinguishing differentiated or mature cells are located above the â€˜â€˜moderately'â€˜pleomorphicmitochon in these lines. The extent of the Golgi complex in the cell dnion (Fig. 3C). â€˜â€˜Extensive'â€˜pleomorphismis characterized by section was estimated according to the number of dictyosomes grossly altered mitochondnial membranes, matnicial myebinfig and their associated vesicles. The Golgi complex in a cell ures (arrows), matnicial vacuoles (V), and distorted shape (Fig. section was considered slightly developed if few dictyosomes 3D). Cnistaeoriented parallel to the longitudinal axis of a giant were present, moderately developed if several dictyosomes or hypertmophied mitochondnion is also depicted in Fig. 3D. occupied regions of the peninuclear cytoplasm, and extensively Due to the variations in the plane through which each mito developed if numerous dictyosomes filled most of the cyto chondnion was sectioned, each section was classified accord plasm. Since profiles of SER and RER are extended throughout ing to the most clearly seen pleomorphic mitochondnion. the cytoplasm, the percentage of observed cytoplasm occupied The accumulated data on the ultrastructurab features of the by these organelles was roughly estimated by dividing the cell mitochondnia are summarized in Chart 3. Little onno mitochon section into fifths beginning on one side of the cell at its longest dnial pleomorphism was observed in those lines derived from axis and proceeding along this axis to the opposite cell side normal tissues. At least some sections from the 3 cell lines making the following cytoplasmic partitions: (a) submembrane, derived from areas peripheral to carcinomas had mitochondria (b) middle, (C) peninucleam; (d) middle, and (e) submembrane. with a moderate degree of pleomorphism, while almost every A cell was scored â€œslight'â€˜ifless than 20% of the cytoplasmic cell section of the tumor-derived lines had mitochondnia show @ area was occupied, â€˜moderate'â€˜if20 to 40% was occupied, ing moderate to extensive pleomorphism. In all cases where and â€œextensiveâ€•ifmore than 40% of the area was occupied. mitochondnial pleomorphism was observed, normal mitochon These characteristics are illustrated in Fig. 2, which shows a dna were also visible. Most of the tumor derived lines, but none cell with extensively developed profiles of SER, a slightly of the nonmalignant lines, had at least some cell sections developed Golgi and no RER (Fig. 2A); moderate Golgi devel showing 2 addition types of abemrancies,hypertrophied mito opment and intermediate degrees of SER and RER (Fig. 2B); chondnia (Fig. 3D), and mitochondnia in which the cnistae and extensively developed RER with no Golgi or SER (Fig. 2C). appear parallel rather than perpendicular to the long axis of All of the epithelial lines, both malignant and nonmalignant, the mitochondrion (Fig. 3D). The remaining mitochondmial display slight to moderately developed Golgi, SER, and RER markers (matnicial granules, ebectmon-lucentand opaque mat characteristic of differentiation and mature cell function (Chart rices) that were scored did not show consistent differences 2). The presence of casein micelles was noted in the breast between the normal and malignant cell lines (data not shown). carcinoma line 578T (24) and an intracytoplasmic canalicular Cytoskeleton. Since all eukanyotic cells seem to contain system typical of stomach parietab cells (49) found in 746T. filaments of 10-nm diameter (tonofilaments), filaments of about Weibel-Palade bodies (19) were not observed in any of the 7-nm diameter, thought to be fibrous actin, and microtubules lines. All of the cell lines were found to be nonendothelial by of variable lengths (6, 16), the cell lines were examined for the platelet adherence test.4 The 2 fibroblastic cell lines, HeLu these 3 structures. Tonofibnils were scored only if they were organized into peninuclear bundles (Fig. 4A). Tonofibnils asso ciated with desmosomes or scattered throughout the cyto 3 The abbreviations used are: SER, smooth endoplasmic reticulum; RER, rough endoplasmic reticulum. plasm, as well as submembranous accumulations of dispersed 4 H. S. Smith, personal communication. microfilaments (Fig. 4A), were not scored in this study. A cell

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1980 American Association for Cancer Research. E. L. Springer section was considered positive for both microfilaments and tochondria and hypertrophied mitochondria were observed to microtubules when the former occurred as discrete bundles gether in all the tumor lines, except 766T and 578T, while both (stress fibers) beneath the plasma membrane (Fig. 46) and the markers were absent from the nonmalignant lines. Mitochon latter was located in the cytoplasm. drial pleomorphism was expressed slightly by the normal lines Chart 4 summarizes the results comparing the nonmalignant and to a moderate degree by the lines derived from nonmalig and tumor-derived cell lines. Both normal and malignant cell nant tissues of cancerous organs. Tonofibril bundles were lines had some sections showing microfilament bundles. In absent in tumor-derived cell lines, while they were frequently contrast, bundles of tonofilaments were absent in tumor-de observed in nonmalignant lines, they were not consistently rived cell lines; however, while they were frequently observed present. Although malignant and nonmalignant cell lines could in the nonmalignant lines, they were not consistently present. not be distinguished using microfilament bundles or microtu Microtubules were rarely observed in any of the epithelial cell bules as markers, this may be due to insufficient cell spread or lines. Two lines of normal fibroblasts, HeLu and 27F, are to the lability of these structures to preparative procedures included in Chart 4 as controls to illustrate that these cell lines inasmuch as the fixation of the cells was performed at room did have microtubules; however, preservation of microtubules temperature and included osmification. However, there is con in these epithelial cells may be labile to the preparative proce flicting evidence concerning the dispersive effect of OsO4 on dures or the cells may be poorly spread, making the observa microfilament bundles (47) and the temperature-dependent tion of microtubules in thin section difficult. depolymerization of microtubules (7, 26) in intact cells. Lastly, To determine whether any of the ultrastructural differences desmosomes, tight junctions, and a similar degree of differen between tumor and nonmalignant cells varied as a function of tiation were found in malignant and nonmalignant lines. growth state, one normal line, 680lnt, and one tumor line, All of the markers present in the carcinoma-derived cells 766T, were compared under confluent and subconfluent cul have been reported to be present in cells in other physiological ture conditions. While microvilli on some cultured normal cells states (19). For example, pleomorphic mitochondria are found decreased in number or disappear as culture density increases in normal fibroblasts (Wl 38) (34); in hepatocytes (49); in virus- (5, 15, 36, 48), no significant difference ( p ~ 0.37) was found transformed (35), carcinoma-derived and sarcoma-derived cell in the number of microvilli-free cells when confluent and sub- lines (54); as well as in a variety of tumors (19) including those confluent 680lnt cultures were compared (Chart 5). However, of mesenchymal origin: osteogenic sarcoma (20), chondrosarcoma (12), giant cell (58), and Ewing's sarcoma (17) bone the 766T tumor cells, like other transformed cells in culture (5, 15, 36), remained villous regardless of culture density. No tumors and fibromyxosarcoma (32). Extremely pleomorphic other ultrastructural characteristics were found to be influ and normal mitochondria have been designated by Tandler er enced by increased culture density when subconfleunt cultures al. (61) to represent the 2 extremes of a continuum in the of 680lnt and 766T were compared to their confluent counter development of the mature oncocyte. Extremely pleomorphic parts except for a higher incidence (p < 0.001) of tight junc mitochondria have been observed in oncocytes of oncocyto- mas (2, 3), Warthin's tumor (62), aged salivary gland (60), tions in confluent 680lnt cultures. Both 766T tumor cultures showed more mitochondrial pleomorphism (p < 0.005), fewer renal adenocarcinoma (30), renal clear cell carcinoma (53), microfilament bundles (p < 0.005), and fewer perinuclear papillary and microfollicular carcinoma of the thyroid gland tonofibril bundles (p < 0.001) than did the 680lnt cultures (63), and breast fibroadenoma (1 ). Hypertrophied mitochondria (Charts 6 and 7). In addition, a tumor arising in an Â¡mmunosup- have been found in the myometrium of pregnancy (11 ) and in pressed mouse after inoculation of 766T cells was also studied fatigued myocardium (45); however, they have been observed and compared to the confluent and subconfluent 766T cul in hepatic tissue surrounding a carcinoma (21), erythroleuke- tures. The mouse tumor in comparison to the confluent 766T mia (22), leukemia (10), fibromyxosarcoma (32), chondrosar- culture had more profiles of RER (moderate versus extensive, coma (12), and osteosarcoma (20). Cristae parallel to the long p < 0.001) and fewer profiles of Golgi (not visible and slight axis have been seen in the mitochondria of the normal adrenal versus moderate, p < 0.005). Aside from the presence of more cortex (33), regenerating axons (31 ), ovarian carcinomas (19), tight junctions than desmosomes and fewer SER profiles (p < and Warthin's tumor (62). Few 10-nm tonofilaments have been 0.025) in the mouse tumor, no other ultrastructural character observed in cancerous basal cells (37), cultured cervical in- istics distinguished the mouse tumor from the confluent and traepithelial neoplastic cells (67), and SV40-transformed 3T3 subconfluent 766T cultures. Lastly, the 766T mouse tumor cells (38); however, numerous tonofilaments have been found expressed the same morphological markers that distinguished in SV40-3T3 revertants and 3T3 cells (38). Defective microtu- the 766T cultures from the 680lnt cultures. bule assembly has been associated with virus-transformed cells (15,18) and Chediak-Higashi polymorpholeukocytes (42). DISCUSSION Reduced numbers of actin bundles have been observed in virus-transformed cells (23, 46), confluent cultures of early- In this paper, the electron microscope was utilized to evalu passage (<26) WI38 cells (34), and subconfluent cultures of ate the cytoplasmic ultrastructure of human epithelial cell lines normal skin fibroblasts (9), while increased numbers of actin derived from normal, nonmalignant tissues of cancerous or bundles have been found in invasive basal carcinoma cells of gans and carcinomas. Consistent ultrastructural markers dis the skin (37), confluent cultures of normal skin fibroblasts (9), tinguished each epithelial category. Mitochondria with longi and 3T3 cells (38), as well as confluent cultures of late-passage tudinal cristal arrangement were found in all the malignant (>40) Wl 38 cells (34). Desmosomes have been reported to be lines, but not in any of the lines derived from tissue peripheral present in carcinoma cell lines (16, 54) and increase in num to tumors or from normal tissue. Extensively pleomorphic mi bers in keratoacanthomas (19), while decreasing in cervical

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1980 American Association for Cancer Research. Cytoplasmic Organelles in Malignant and Nonmalignant Cell Lines carcinoma cells (39) and epitheliotopic viral infections (8). Tight mammary epithelial cells in nipple aspirates, short- and long junctions, but not desmosomes, have been observed in sam term culture, we found similar but not identical nuclear (56) coma-derived cell lines (54). While a few poorly formed des and cytoplasmic changes which were consistent for histopath mosomes on atypical tight junctions have been found in undif ological type both in vivo and in vitro. Likewise, studies corn fementiated mesenchymal cell lines (16); osteosancoma (20) paring 10 primary cultures of human mammary epithelial cells and chondrosarcoma (12) tumors are deficient in these stnuc derived from normal tissue, nonmalignant tissue peripheral to tunes. carcinoma, and carcinomas showed that moderate mitochon In view of the fact that these markers are present in cells in dmialpleomonphism was common in cells derived both from a wide variety of physiological states, it is surprising that, in tissue peripheral to carcinoma and from breast carcinomas, this and the previous study on nuclear markers (56), consistent while cells derived from normal tissue showed no or only slight differences appear to exist between the malignant and non pleomomphism.Tonofilament bundles and microfilaments were malignant cells in culture. In both studies, great care was taken found in cells derived from all sources; however, fewer were to ensure objectivity. Each cell line was scored by a single observed in carcinoma-derived cells.5 operator unaware of the origin of the specimen, and every cell The interpretation of these observations must be made with section on the grid was evaluated by determining the degree of caution, however; this system does offer quantitative and qual marker expression. Many reports from in vivo observations of itative ultrastructural criteria with the potential for assessing tumors (19) have stated theme is no consistent pattern of transformation in vitro, especially the effect of carcinogens on characteristics to distinguish normal from malignant cells. How normal human epithelial cells. In addition, these studies may ever, in the only (to my knowledge) in vivo semiquantitative provide insights into the mechanism of malignant progression. ultrastructumal studies (27, 28), noncoded specimens of liver While it is unlikely that the observed morphological changes from tumor-beaming matsand transplantable hepatomas were represent specific functions associated with malignant trans compared. While the hepatomas did not consistently express formation, the total of all the changes may reflect a common similar ultrastructumalalterations, they were distinguished from metabolic state that is the foundation for carcinoma induction. hepatocytes by possessing to a greater degree the following Themehavebeen a few reports (13, 66) at the light microscopic characteristics: nuclear envelope dilation: cytoplasmic invagi level indicating that macroscopically normal tissue peripheral nations of the nucleus; large and multiple nucleoli with micro to carcinoma contains numerous foci of atypical hypemplasias segregated components; mitochondnia with longitudinal cris suggesting that the entire organ is progressing toward malig tae; and a large Golgi complex. Although there are no human nant transformation. Consequently, those ultmastmuctumal hepatoma cell lines in this study, many of the malignant cell markers found in varying degrees in cells of lines derived from lines express these nuclear (56) and cytoplasmic characters tissue peripheral to carcinoma and to a greater extent in exclusively or to a greaten degree than do those lines derived carcinoma derived cells may reflect metabolic changes inher from tissue peripheral to carcinomas or from normal tissue. ent in early pmemalignancy.Therefore, biochemical studies on While no consistent ultrastructumaldifferences have been seen nucleoli and nuclear envelopes (56), mitochondnia, and 10-nm after transformation of cells in culture (16), many studies of tonofilaments may be important in elucidating the nature of the camcinogenesishave used rodent cell lines which have a high ultrastructumaldifferences in these cell components. incidence of spontaneous transformation (50) and may already Finally, these studies suggest the need for the development be transformed prior to the addition of a chemical carcinogen of additional human epithelial cell lines to determine which or oncogenic virus (51). For example, the rodent cell line 3T3 ultrastructural characters are omganotypic. In view of the suc ultrastructurally resembles the SV4O transformant (38) and is cess in initiating human epithelial cell lines in this and other similar to the tumor cells described in this study. However, 3T3 laboratories (16), this is a feasible approach. In addition, since has recently been suggested to be poised in the premalignant the growth properties of epithelial cells are different from those state (4). Although no normal cell lines were presented for of fibroblasts (64) and the morphological criteria for in vitro comparison, Seman and Dmochowski (54) have found human transformation have been derived mainly from fibmoblasts(25), tumor cells that ultmastmuctumallyresemblethe tumor cells de it is important to develop new quantitative systems for assaying scribed here. Advances in identifying differences between tu the transformation of epithelial cells in culture. momand normal cells have been thwarted by the lack of normal cells cultured under conditions identical to those for tumor cells ACKNOWLEDGMENTS (16) and the paucity of systematic comparisons of tumor and normalcellsinvivo(19). The author gratefully acknowledges the helpful comments and suggestions of Drs. Mina Bissel, Gertrude Forte, Adeline Hackett, Thomas Hayes, Walter Nelson Since these cell lines have been derived from different or Rees, and Helene Smith during the preparation of this manuscript; and Marie gans, it is difficult to separate organotypic from malignant Mizelle and Alan Hiller for excellent technical assistance. Special thanks are characters. In particular, all of the lines derived from nonmalig extended to Dr. Lucian LeCam, Department of Statistics, University of California, Berkeley, for the statistical evaluations. nant tissues of a cancerous organ are kidney, and 3 of the normal lines, while derived from intestine at roughly the same stage of in utero development, are fetal, which introduces REFERENCES another set of parameters into the interpretation of the obser 1. Archer, F., and Omar, M. Pink cell (oncocytic) metaplasia in a fibro-adenoma vations. However, 2 of the tumor-derived lines, 761T and 769T, of the human breast. Electron microscopic observations. J. Pathol., 99: and one cell line derived from normal bladder, 76761 , repre 119â€”127,1969. sent the same cell type. In addition, in preliminary studies (57)

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Scanning electron microscopy of in vitro C.), 188: 68-70, 1975. chemically transformed mouse embryo cells. J. Cell Biol., 68: 654-664, 5. Borek, C., and Fengolio, C. M. Scanning electron microscopy of surface 1976. features of hamster embryo cells transformed in vitro by X-irradlatlon. 37. McNutt, N. S. Ultrastructural comparison of the Interface between epithellum Cancer Res., 36: 1325-1 334, 1976. and stroma in basal cell carcinoma and control human skin. Lab. Invest., 35: 6. Buckley, I. K., and Porter, K. R. Cytoplasmic fibrils in living cultured cells. A 132â€”142,1976. light and electron microscopic study. Protoplasma, 64: 349-380, 1967. 38. McNutt, N. S., CuIp, L. A., and Black, P. H. Contact revertant cell lines 7. Brinkley, B. R., and Cartwright, J. Cold labile and cold stable microtubules isolatedfromSV4Otransformedcells.J. CellBlol.,56: 412â€”428,1973. in themitoticspindleofmammaliancells.Ann.N.Y.Acad.Sci.,253:428â€” 39. McNutt, N. S., and Weinstein, R. S. Carcinoma of the cervix: deficiency of 439, 1975. nexus intercellular junctions. Science (Wash. D. C.), 165: 597â€”599,1969. 8. Cheville, N. F. Cell Pathology. Ames, Iowa: The Iowa State University Press! 40. Montesano, R., St. Vincent, L. C., Devron, C., and Tomatis, L. Production of Ames, 1976. epithelial and mesenchymal tumors with rat liver cells transformed in vitro. 9. Comings, D. E., and Okada, T. A. Electron microscopy of human fibroblasts Int. J. Cancer, 16: 550â€”558,1975. in tissue culture during logarithmic and confluent stages of growth. Exp. Cell 41. Namba, M., Masuji, H., and Sato, J. Carcinogenesis in tissue culture. ix. Res., 61: 295-301, 1970. Malignant transformation of cultured rat cells treated with 4-nitroquinoline 10. De Harven, E. Human leukemic cells in tissue culture: an electron micro 1-oxide. Jpn. J. Exp. Med., 39: 253â€”265,1969. scope survey. Cancer Res., 2 7: 2447-2458, 1967. 42. Oliver, J. M. Defects in cyclic GMP generation and microtubule assembly in 11. Dessouky, D. A. Electron microscopic studies of the myometrium of the Chediak-Higashiandmalignantcells.In: M. BorgerandM. DeBrabander guinea pig. Am. J. Obstet. Gynecol., 100: 30â€”43,1968. (eds.), The International Symposium on Microtubules and Microtubule Inhib 12. Erlandson, R. A. , and Huvos, A. G. Chondrosarcoma: Alight and electron itors, pp. 341-354. New York: American Elsevier Publishing Co., 1975. microscopic study. Cancer (Phila.), 34: 1642â€”1652,1974. 43. Owen, D. B. Handbook of Statistical Tables. Reading, Mass.: Addison 13. Farrow, G. M., Utz, 0. C., and Rife, D. D. Morphological and clinical Wesley Publishing Co., 1962. observations of patients with early bladder cancer treated with total cystec 44. Owens, R. B., Smith, H. S., Nelson-Rees,W. A., and Springer, E. L. Epithelial tomy. Cancer Res., 36: 2495â€”2501,1976. cell cultures from normal and cancerous human tissues. J. NatI. Cancer 14. Fedoroff, S. Proper usage of animal tissue culture terms. J. NatI. Cancer Inst., 56: 843-849, 1976. Inst., 38: 607-61 1, 1967. 45. Pelosi, G., and Agliati, G. The heart muscle in functional overioad and 15. Fonte, V., and Porter, K. R. Topographic changes associated with the viral hypoxia. A biochemical and ultrastructural study. Lab. Invest., 18: 86â€”93, transformation of normal cells to tumorigenicity. In: Eighth International 1968. Congress on Electron Microscopy (Australian Academy of Science, Can 46. Pollack, R., Osborn, M., and Weber, K. Patterns of organization of actin and berra), pp. 334-335, 1974. myosin in normal and transformed cultured cells. Proc. NatI. Acad. Sci. U. 16. Franks, L. M., and Wilson, P. D. Origin and ultrastrucutre of cells in vitro. Int. S. A., 72: 994-998, 1975. Rev. Cytol., 48: 55â€”139,1977. 47. Pollard, T. D., and Maupen, P. Electron microscopy of cytoplasmic contrac 17. Friedman, B., and Gold, H. ultrastructure of Ewings sarcoma of bone. tile proteins. In: Ninth International Congress on Electron Microscopy, To Cancer (Phila.), 22: 307-322, 1968. ronto. Canada: Microscopical Society of canada, Vol. 3, pp. 606â€”614, 18. Fuller, G. M., and Brinkley, B. R. Structure and control of assembly of 1978. cytoplasmic microtubules in normal and transformed cells. J. Supramol. 48. Porter, K. R., Prescott, D., and Frye, J. Changes in surface morphology of Struct., 5: 497â€”514,1976. Chinese hamster ovary cells during the cell cycle. J. Cell Biol., 57: 815â€” 19. Ghadially, F. N. Ultrastructural Pathology of the Cell. Boston: Butterworths, 836, 1973. 1975. 49. Sandborn, E. B. Cells and Tissues by Light and Electron Microscopy, Vol. 1. 20. Ghadially, F. N., and Mehta, P. N. Ultrastructure of osteogenic sarcoma. New York: Academic Press, Inc., 1970. Cancer (Phila.), 25: 1457â€”1467,1970. 50. Sanford, K. K. â€œSpontaneousâ€•neoplastictransformation in vitro. In: R. L. 21. Ghadially, F. N., and Parry, E. Ultrastructure of human hepatocellular car Clark,R.W.Cumley,andJ.E.McCay(eds.),TheTenthInternationalCancer cinoma and surrounding non-neoplastic liver. Cancer (Phila.), 19: 1989â€” Congress, Vol. 1, pp. 76â€”82.Chicago:Year Book Medical Pubi. Inc., 1970. 2004, 1976. 51. Sanford, K. K., Barker, B. E., Woods, M. W., Parshad, R., and Law, L. W. @ 22. Ghadially,F. N., and Skinnider,L. F. Giantmitochondriainerythroleukemia. Searchfor Indicators'of neoplasticconversionin vitro.J. NatI.Cancer J. Pathol., 113â€”124,1974. Inst., 39: 705-733, 1967. 23. Goldman, R. D., Yerna, M-J., and Schloss, J. A. Localization and organize 52. Sato, J., Namba, M., Usui, K., and Nagano, D. Carcinogenesis in tissue tion of microfilaments and related proteins in normal and transformed cells. culture. vlll. Spontaneous malignant transformation of rat liver cells In long J. Supramol. Struct., 5: 155â€”183,1976. term culture. Jpn. J. Exp. Med., 38: 105â€”118,1968. 24. Hackett, A. J., Smith, H. S., Springer, E. L., Owens, R. B., Nelson-Rees, W. 53. Seljelid, R., and Ericsson, J. I. E. An electron microscopic study of mito A., Riggs, J. L., and Gardner, M. Two syngeneic cell lines from human chondria in renal cell carcinoma. J. Microscopie, 4: 759â€”770,1965. breast tissues. One aneuploid mammaryepithelial (H5578T) and one diploid 54. Seman, G., and Dmochowskl,L Ultrastructuralcharacteristicsof human myoepithelial (578Bst). J. NatI. Cancer Inst., 58: 1795â€”1806,1977. tumor cells in vitro. In: J. Fogh (ed.), Human Tumor Cells in Vitro. pp. 395â€” 25. Handleman, S. L., Sanford, K. K., Tarone, R. E., and Parshad, R. Cytology 486. New York:PlenumPress, 1975. of spontaneous neoplastic transformation in culture. In Vitro, 13: 526â€”536, 55. Smith, H. S., Owens, R. B., Nelson-Rees, W. A., Springer, E. L., Dollbaum, 1977. C. M., Hackett, A. J., and Williams, R. Epithelial cell cultures from human 26. Hayat, M. A. Principles and Techniques of Electron Microscopy, vol. 1. New carcinomas. In: H. E. Neiburgs (ed), The Third International Symposium on York: von Nostrand Reinhold Co., 1970. the Detection and Prevention of Cancer (1976), Vol. 2, pp. 1465â€”1479. 27. Hruban, Z., Mochizuki, Y., Slesers, A., and Morris, H. P. A comparative New York: Marcel Dekker, 1978. study of cellular organelles in Morris hepatomas. Cancer Res., 32: 853â€” 56. Smith, H. S., Springer, E. L., and Hackett, A. J. Nuclear ultrastructure of 867, 1972. epithelial cell lines derived from human carcinomas and non-malignant 28. Hruban, Z., Swift, H., and Rechcigi, M. Fine structure of transplantable tissues. Cancer Res., 39: 332â€”344,1979. hepatomas of the rat. J. Natl. Cancer Inst., 35: 459-595, 1965. 57. Springer, E. L., Hackett, A. J., and Stampfer, M. R. Surface topography and 29. Katsuta, H., and Takaoka, T. Carcinogenesis in tissue culture xlv: Malignant ultrastructure of normal, benign, atypical and malignant human mammary transformation of rat liver parenchymal cells treated with 4-nitroguinoline 1- epithelial cells. J. Electron Micros., 26: 176, 1977. oxide in tissue culture. J. NatI. Cancer Inst., 49: 1563â€”1576,1972. 58. Steiner, G. C.. Ghosh, L., and Dorfman, H. D. Ultrastructure of giant cell 30. Keyhani, E. Anomalies de structure de mitochondries dans un adenocarci tumor of bone. Human Pathol., 3: 569-586, 1972. nome renal spontanÃ©deIa souris. Arch. Biol. (Liege), 80: 153â€”162,1969. 59. Straehelin, L. A. Structure and function of intercellular junctions. Int. Rev. 31. Lampert, P. W. A comparative electron microscopic study of reactive, Cytol., 39: 191â€”283,1974. degenerating, regenerating and dystrophic axons. J. Neuropathol. Exp. 60. Tandler, B. Fine structure of oncocytes In human salivary glands. Virchow's Neurol., 26: 345-358, 1967. Arch. Pathol. Anat., 341: 317-331 , 1966. 32. Leak, L. V., Caulfield, J. B., Burke, J. F., and McKhann, C. F. Electron 61. Tandler, B., Huller, R. V., and Erlandson, R. A. Ultrastructure of oncocytoma microscopic studies of a human fibromyosarcoma. Cancer Res., 2 7: 261â€” of the parotid gland. Lab. Invest., 23: 567â€”580,1970. 285, 1967. 62. Tandler, B.. and Shipkey, F. H. Ultrastructure of Warthin's tumor. I. Mite 33. Lever, J. D. Physiologically induced changes in adrenocortical mitochondria. chondria. J. Ultrastruct. Res., 11: 292â€”305,1964.

808 CANCERRESEARCHVOL. 40 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1980 American Association for Cancer Research. Cytoplasmic Organelles in Malignant and Nonmalignant Cell Lines

63. Tonietti, G., Baschieri, L., and Salabe, G. Papillary and foilicular carcinoma 1975. of human thyroid. An ultrastructural study. Arch. Pathol., 84: 601 â€”613, 66. Welllngs, S. R., Jensen, H. M., and Marcum, R. G. An atlas of subgross 1967. pathology of the human breast with special reference to possible precan 64. Weinstein, I. B., Orenstrien, J. M., Gebert, R., Kaighn, M. E., and Standler, cerous lesions. J. NatI. Cancer Inst., 35: 231 â€”273,1975. U. C. Growth and structural properties of epithelial cell cultures established 67. Wilbanks, G. D. In vitro studies on human cervical epithelium, benign, and from normal rat liver and chemically induced hepatomas. Cancer Res., 35: neoplastic. Am. J. Obstet. Gynecol., 121: 771â€”788,1975. 253â€”263, 1975. 68. Williams, G. M., Weisburger, E. K., and Weisburger, J. H. Isolation and long 65. Weinstein, I. B., Vamaguchi, N., Orenstein, J. N., Gabert, R., and Kaighn, M. term culture of epithelial cells from rat liver. Exp. Cell Res., 69: 106â€”112, E. In: L. E. Gerschenson and E. B. Thompson (eds.), Gene Expression and 1971. Carcinogenesis in Cultured Liver, p. 441 . New York: Academic Press, Inc.,

ULTRASTRUCTUREOFTHE PLASMA MEMBRANE

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MARCH 1980 809

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810 CANCER:, RESEARCH VOL. 40

MITOCHONDRIA!. ULTRASTRUCTURE

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MARCH 1980 811

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'Primary unknown Chart 4. Summary of observations on microfilament bundles, tonofibril bundles, and microtubules. Trans., transitional.

812 CANCER RESEARCH VOL. 40

Cytoplasmic Organe!!es in Ma!ignant and Nonmalignant Cell Lines

ULTRASTRUCTURAL COMPARISON OF NORMAL AND MALIGNANT CELLS IN VARIOUS GROWTh STATES

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Fig. 2. Cells illustrating degrees of difterentiation expressed by Golgi (G), SER (S), and RER (R) development. A, well-differentiated cell showing â€œextensiveâ€• profiles of SER (S), â€˜slight'statusfor Golgi (G) development, and â€˜notvisibleâ€•statusfor RER; tumor-derived cell line 675T. 8, sIghtly differentiated cell showing @ . moderate' â€˜statusfor Golgi (G) development and slightâ€•statusfor SER (S) and RER (R); normal fetal Intestine cell line 6801nt. C, well-differentiated cell showing @ . extensive' â€˜RER CR) development, not visibleâ€• status for SER. and â€˜slight' â€˜Golgi (G) development; tumor-derived cell line 696T. Marker, 1 tim. Uranyl acetate-lead citrate.

814 CANCERRESEARCHVOL. 40

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Fig. 4. Arrangements of filamentous structures in the cytoplasm. In A, dispersed microfilaments (M) are located beneath the plasma membrane, and tonofibril bundles(TB)surroundthenucleusof the normalfetal intestinecellsof 68Olnt.B, microfilaments(M)arrangedin an electron-densebundlebeneaththeplasma membrane; tumor-derived cell line 578T. Marker, 0.5 tim. Uranyl acetate-lead citrate. 817

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1980 American Association for Cancer Research. Comparative Study of the Cytoplasmic Organelles of Epithelial Cell Lines Derived from Hu man Carcinomas and Nonmalignant Tissues

E. Louise Springer

Cancer Res 1980;40:803-817.

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