- Email: [email protected]
Ultrastructural analysis of malignant conversion in vitro of epithelial cells derived from normal adult rat liver
Europ. J. Cancer Vol. 12, pp. 527-534. Pergamon Press 1976. Printed in Great Britain
Ultrastructural Analysis of Malignant Conversion In Vitro of Epithelial Cells Derived from Normal Adult Rat Liver* S. KARASAKI~$, A. SIMARDJ" and G. DE LAMIRANDE§ $ Institut du Cancer de Montrdal, Centre Hospitalier Notre-Dame, and ~Ddpartement d'Anatomie et §Ddpartement de Biochimie, Universitd de Montrdal, Montrgal, Canada Abstract A tumorigenic cell line of epithelial-type was establishedfrom a long-term culture of isolated liver cells from a normal adult Wistar rat. Electron microscopic examinations of foci of transformed epithelial cells in the primary culture revealed production of virus particles in association with membranes of the nuclear envelope, endoplasmic reticulum and cell surface. The epithelial cells were found in quiescent state and had intranuclear canalicular membranes. By trypsinization, the cells were initiated into a rapid replication and showed subcellular differentiation. Annulate lamellae and paired cisternae emerged in passage 1. Viral production ceased at this stage. Close apposition of adjacent cells became prominent in passage 9, when the cell line first revealed tumorigenicity upon transplantation into neonatal Wistar rats. Concomitantly, cytochemical tests gave a positive reaction of A TPase activity on the plasma membranes. Surface A TPase activity continued to show in the epithelial cells following passages in vitro as well as in tumor cells in vivo. The possible origin of epithelial cells in vitro from hepatocytes was suggested by the demonstration of peroxisomes and bile canalicular-like structures in the carcinoma cells.
of liver cells [8, 17, 26]. O f these reports, Weinstein et al. [8, 26] have focused on the presence of oncorna virus particles in rat liver cell lines and their correlation with transformed cells. The sequential process of in vitro neoplastic transformation is very little understood at the subcellular level. This paper reports the establishment and morphological characterization of a tumorigenic cell line of epithelial-type, derived from the liver of a normal adult rat. The studies of cells in the primary culture and subcultures involve fine structural and cytochemical observations with the objective of obtaining clues to the nature and sequence ofsubcellular events which may constitute cytodifferentiation toward malignant conversion in vitro. The carcinomas, produced by the implantation of cultured cells in rats, are ultrastructurally examined in special attention to cytochemical properties of differentiated hepatocytes.
INTRODUCTION ISOLATED hepatocytes from normal rat liver usually have a limited life span in vitro [1-4]. Under certain conditions, however, liver cells m a y continue to multiply [5-12] and eventually become malignant during years of long term cultivation [13-15]. Malignant conversion in vitro of cultured liver cells has been induced or accelerated by treatments with chemical carcinogens [8, 16-19] or oncorna viruses [20]. The detection of malignant conversion in vitro of epithelial cells is particularly difficult, since little alteration is induced in cell morphology at the critical step [21]. Although m a n y studies were performed on hepatocarcinogenesis in vivo [22-25], there are few reports of subcellular analyses on in vitro transformation
Accepted 26 January 1976 *This work was supported by grants from the National Cancer Institute of Canada, le Minist~re des Affaires sociales du Q.udbec, La Fondation J. H. Biermans and Les Fondations J. Rhdaume. ++Research Associate of the National Cancer Institute of Canada.
MATERIAL AND METHODS Cell separation Rats of the Wistar strain were obtained from 527
528
S. Karasaki et al.
Canadian Breeders, Inc., St. Constant, Que., Canada. The cell line studied here was derived from the liver of a normal male weighing 200 g. The liver was dissociated on M a y 10, 1973. The cell separation technique employed was essentially the same as that described by Berry and Friend [3]. After perfusion through the hepatic vein with Ca2+-deficient Hank's solution containing 0.05% hyaluronidase (Sigma) and 0.025% collagenase II (Sigma), liver ceils were suspended in a mixture (1:1) of Ham's F12 culture medium (GIBCO) and of Hank's solution containing 0-5 M sucrose and passed through two successive sieves with 250 and 40/l mesh. The enzymatic dissociation of an adult rat liver yielded 2"5 x 10 s cells. The cell population consisted almost exclusively of hepatocytes (99.1%) as determined with Carnoy-fixed smears stained with hematoxylin-eosin. Cell culture
The standard culture medium was H a m ' s F12 containing 100 units of penicillin G and 100 pg of streptomycin per ml and, supplemented with 5 or 10°/O fetal calf serum (GIBCO). Isolated hepatocytes were plated at 37°C in plastic T-flasks ( 2 5 c m 2, Falcon). Cellular concentration was 5 x l 0 6 per 10 ml. Within 6 hr of explanting in a plastic flask, a majority of spherical hepatocytes were packed in a layer of aggregates. They had an angular outline and were loosely attached to the plastic surface during the 1st week period. The medium was partially changed 2 or 3 times a week and were maintained in the same flasks from 4 months to more than 18 months in some cases. Regular determination of cell numbers in flasks showed little variation or a very slow decrease. Six months after explantation, 7 separate cultures showed signs of cell growth. O n J a n u a r y 4, 1974, the first subculture was performed on one of the flasks. To dislodge the cells, a mechanical dispersion method was applied after 10rain of treatment with a 0.25% trypsin solution at 37°C. Further passages in vitro were continuously done at 5-8 days intervals with trypsinization. Plating efficiency in earlier passages was less than 5% of the cells. In passage 4, cells grew well in monolayer (Fig. 1). In passage 6 or later, the cells were easily detachable by trypsinization. In passage 12, the logarithmic growth was readily resumed after a lag period of 18 to 24 hr. At this point, the plating efficiency was in excess of 20% and the population doubling time was less than 30 hr in the
4,
°-4
/,,-'"
-12-
Fig. I. Growth patterns of cultured liver cells at the 4th ( - - O - -) and 12 th ( - - 0 - - ) passages. Cells were placed in a series of T-5 flasks (75 cm 2) to each of which 15 ml of Ham's F12 mediumplus 10% fetal bovine serum was added. Each day, the culture was trypsinized and the number of cells was measured in Coulter counter.
logarithmic phase (Fig. 1). The saturation of cell density resulted in the inhibition of growth (Fig. 5). More than 90 passages have been done over a period of 20 months. Tumorigenicity
Newborn Wistar rats (6-18 hr after birth) were used for tumorigenicity tests of the cultured cells. Cells were dissociated by either trypsinization or mechanical dispersion. A suspension o f l 0 6 to 2 x 106 cells in 0.05-0.1 ml of culture medium was injected subcutaneously or intraperitoneally. When transplanted either i.p. or s.c. into newborn rats with 2 x 106 cells from passage 6, no tumors were formed (Table 1). In the same batch of animals, positive results were obtained on s.c. transplantation of an established cell line, R L C [4, 27]. 2 x 106 cells of passage 9 could produce tumors on i.p. transplantation. Further passages developed tumors on s.c. transplantation of 1-2 x 106 cells with a latency period of 10-18 days (Table 1). Some of the tumors were explanted and grown in vitro. A s.c. inoculation of 106 cells gave a solid tumor of 10 cm 3 volume after 4 weeks and killed the recipient. I.p. transplants developed into many solid tumors on the surface of peritoneum and the diaphragm. Metastasis into the eye was found in an i.p. transplant. Routine histologic examinations were done on Carnoy-fixed, paraffin-embedded and toluidine blue-stained tissues. Electron microscopy
The cells in plastic culture flasks were fixed in situ for 30 min to 1 hr with 2.5% glutar-
Fig. 11. A TPase reaction in glutaraldehyde-fixed tumor section. A positive reaction on the pattern of lead phosphate precipitates follows the course of the plasma membranes (arrows). Intranuclear canaliculi ( NC). x 12,000. Fig. 12. Diaminobenzidine-H20 2 reaction in glutaraldehyde-fixed tumor section. Peroxidase activity is seen located in microbodies of carcinoma cells (arrow). x 30,000.
Fig. 9. Photomicrograph of a solid tumor produced by i.p. transplantation of the cultured cells in passage 9. The peripheral portion of a solid tumor shows three parts with different histological patterns as well as variation in staining characteristics. Fibro-stromal tissues (S) and capillaries (E) of the host. Carnoy-toluidine blue. x 225. Fig. I O. Electron micrograph of a part of carcinoma tissue. Bile canalicular-like surfaces (arrows). Opaque amorphous material resembling biliary products, x 6,750.
Fig. 7. A TPase reaction in epithelial cells from a preconfluent culture of passage 9. The surface membranes of adjacent cells were seen covered with bands of opaque reaction product. Regional differences in the surface deposits of the reaction product, ranging from few tiny particles to continuous bands, x 7,500. Fig. 8. Ruthenium red staining of epithelial cells from a confluent of passage 20. In tangential section of 3 adjacent cells, the surface membranes are closely apposed and covered with an electron opaque layer of ruthenium red stained coat. Pinocytotic vesicles (arrows). x 15,000.
Fig. 6. (a)-(d).
Rat liver epithelial cells in passage 1.
(a) Paired cisternae (PC) in the cytoplasm. Small A particle within a smooth vesicle (a). Intranuclear canaliculi (arrows). A large nucleolus with prominent granular components. Uniformly distributed chromatin, x 15,000. (b) Perpendicular section of annuli in cytoplasmic annulate lamellae ( AL). Small A particle within a smooth vesicle (a). x 52,500. (c) Tangential section of annuli in cytoplasmic annulate lamellae ( AL). Parallel arrays of rough endoplasmic reticulum cisternae. Large A particles within a smooth vesicle (A). x 30,000. (d) Cytoplasmic annulate lamellae (AL) in continuity with rough endoplasmic reticulum. Large A particle within a smooth vesicle (A). x 22,500.
Fig. 4. Liver epithelial cells in the primary culture (after 7 months). Intercellular and intracisternal virus particles. Budding (1), immature (2) and mature (3)forms of Cparticles. Large A particles in the cisternae of nuclear envelope. The nucleolus (NO) show a decreased amount of granular components which are segregated from fibrillar components, x 30,000. Fig. 5. Liver epithelial cells in the primary culture (after 7 months). Free and budding form~ of intracisternal A particles. Small A particles (a) within the perinuclear eisternae and the vesicles of endoplasmic reticulum. Large A particles (A) within cytoplasmic vacuoles. Intranuclear membranes (arrow) and lipid (L). x 67,500.
Fig. 2. Electron micrographs of rat liver epithelial cells in the primary culture (after 7 months). Intranuclear canaliculi (IN). Virus particles (arrows). Undulated cell surface, x 12,000. Fig. 3. Electron micrographs of rat liver epithelial cells in the primary culture (after 7 months). Large and small nuclei with different patterns of chromatin distribution. Virus particles (arrows). Endoplasmic reticulum with flattened sacs, the content of which are moderately dense, x 22,500. (to face page 528)
Malignant Conoersion: o f L'iver Cells Table 1.
Tumorigenicity of cultured epithelioid cellsfrom rat liver
Days after subculture*
Passage number]"
44 55 65 80 98
--6---9---12-14 --9--C1--1--
118
157 389
529
--20--
--16--C1--2---50--
Tumor incidence + i.p. § s.c. § 0/3 2/3 -2/2 -2/4 ---
0/2 0/4 1/3 2/5 5/6 3/4 2/2 3/4
Average latency period (days) --15 18 12 13 14 10
*Plus 237 days in the primary culture of isolated hepatocytes. ].Passage number in vitro, except C1 (1 passage in vivo). ++Number of animals with carcinoma/Number of newborns inoculated. §Route of implantation of 1-2 x 106 cells per animal. aldehyde (Ladd) in 0.1 M sodium cacodylateHC1 buffer (pH 7.1), washed for 1-4 hr in 7"5% sucrose solution, post-fixed for 1 hr with 1.4% OsO4 in 0.1 Ms-collidine buffer (pH 7.1), dehydrated in a graded ethanol-water series and embedded in Epon. Sections were prepared for both light and electron microscopy. Thin sections were stained with uranyl acetate and lead citrate, and were viewed in a J E M - 7 A electron microscope. Specimens of tumor tissues were similarly processed.
Cytochemistry To detect cytochemical alterations of surface membrane of the cells during passages in vitro and in vivo, the method of Wachstein and Meisel [28] for ATPase demonstration was applied on glutaraldehyde-fixed cells in situ in plastic flasks or on sections of fixed tumors. The method of Novikoff and Goldfisher [27] was similarly applied for detection of peroxisomes [29]. Fixatives containing ruthenium red were also used to identify carbohydratecontaining structures on the cell surface [30]. RESULTS
The primary culture During 6 months period the originally explanted hepatocytes remained attached over the surface of flask bottom. Each of 7 separate cultures from the single preparation ( < 2"5 x 10 a cells) contained 3-10 foci of colonies of epithelial cells and sparsely scattered stellatetype cells. The foci of epithelial cells grew very slowly, although mitoses were never detected. The ultrastructural examinations were performed on the colonies of epithelial cells after
7 and 8 months in culture. It appeared that they had lost almost all of their original tissue characteristics at the subceUular level, but they were found to vary markedly from one another in fine structure. The cross section of epithelial cells in situ showed contacts of the plasma membrane to the plastic substratum and an accumulation of microfilaments and microtubules adjacent to the membrane, suggesting a strong adhesion of cells with the substratum. Cell-to-cell contacts were mostly focal at the crests of surface undulation (Fig. 2). Cells contained more than one nucleus, which demonstrated different patterns of chromatin distribution (Fig. 3). The nuclei were extremely deformed and lobulated in outline, frequently possessing deep cytoplasmic invaginations (Fig. 2-5). The nucleoplasm included canalicular membranes, of which structure appeared to be similar to that of the inner nuclear membrane (Fig. 2). In the cytoplasm, there were membranous profiles of endoplasmic reticulum scattered singly or in parallel arrays (Fig. 2-5). Numerous structures resembling oncorna virus particles were frequently seen (Fig. 2-5). Extracellular C particles [31] and two types ofintracisternal A particles [32] were identified (Fig. 4 and 5). C particles measured 90-100 nm dia and were formed by a typical process of budding from the cell membrane into the intercellular space (Fig. 4). Immature C particles comprised a relatively electronlucent core surrounded by an electron dense shell (Fig. 4). Mature C particles contained a central dense core surrounded by an irregularly contoured membrane (Fig. 4). Small and large types of A particles were present in the cisternae of nuclear envelope and the vesicles of endoplasmic reticulum (Fig. 5). Particle size ranged from 70-80 nm for the small type and
530
S. Karasaki et al.
from 9 0 - 1 1 0 n m for the large type (Fig. 5). Both types of particles appeared to contain 2 concentric dense shells and an electron lucent center (Fig. 5). Budding forms of both types of particles were seen from the m e m b r a n e s of either endoplasmic reticulum or nuclear envelope (Fig. 5). Subcultures T h e cultures of passage 1 contained epithelial cells mixed with stellate-type cells. Mitoses were frequently seen. Following further subcultures, the cells became more homogeneous in size and shape. Comparative studies at the ultrastructural level were performed and the subcellular features in epithelial cells in the p r i m a r y culture as well as in subcultures are summarized in Table 2 with reference to the sequence of passage numbers. In passage 1, the majority of cells showed less irregularities of nuclear fine structure when compared to those of p r i m a r y culture. O n l y a few exhibited intranuclear canalicular membranes (Fig. 6a). Paired cisternae (Fig. 6a) and annulate lamellae (Fig. 6b-d) were frequently found in passage 1. These cytoplasmic m e m b r a n o u s structures morphologically resembling the nuclear membrane were often located adjacent to the nucleus (Fig. 6a). T h e y were also found in continuity with the m e m b r a n e s of endoplasmic reticulum (Fig. 6d). Virus particles were seen within the vesicles of endoplasmic reticulum adjacent to annulate lamellae or paired cisternae (Fig. 6a-d) and rarely in association Table 2.
with the cell surface, but none within the perinuclear cisternae. T h e fine structure of nuclei and cytoplasm did not appear to change remarkably with passage number. There were few specializations of cytoplasmic organelles, accompanying the confluency of cells. R o u g h endoplasmic reticulum cisternae were quite p r o m i n e n t in post-confluent cultures. Peroxisomes and glycogen particles were not detectable by cytochemical methods. Microfilaments adjacent to the plasma membranes were rare at the preconfluency and increased significantly at the confluency. A few virus particles were detectable by passage 6, while later their detection was seldom. In passages 4 and 6, the junctions of close apposition between individual cells were mostly focal and the intracellular dilations were evident. Using the Wachstein-Meisel technique, ATPase reaction was not seen by passage 8 (Table 2). In passage 9, however, some colonies showed the reaction product at the outer surface of plasma membranes of epithelial cells (Fig. 7). T h e distribution of the reaction product varied a m o n g individual cells, such as complete absence from some and its restriction to certain regions on the surface of others (Fig. 7). T h e ATPase-positive cells were ultrastructurally indistinguishable from neighboring cells which showed no activity. All epithelial cells from passage 12 exhibited reaction products at r a n d o m along the entire plasma membranes, although it was more a b u n d a n t on the apposed surfaces t h a n the
Sequentialalterations of cultured epithelial cells after in vitro and in vivo passages Cytochemical reaction]"
Electron microscopic detection]" Passage number*
Virus particles
Intranuclear canalicular membranes
--0---1---4--
+ +/-/+
+ -/+ -/+
--6--
--/+
--8.
.
. 112 . --14 . --9--C1--1---20 . - - 16--C1--2 . --50 .
.
--9
.
.
. . .
. . -/+
Surface close appositions
Surface ATPase activity
-/+ -/+ -/+
----
--/+
--
/+
-/+
. .
.
--
.
-I+ .
. .
-/+ + -/+
--
.
. . .
Cytoplasmic annulate lamellae
. .
*Passage number in vitro, except C1 (1 passage in vivo). I"+ : Frequent detection in thin sections of ceils. +/-- : infrequent, - / + : rare, --: seldom or never encountered.
+ + + + + + +/-
+/+ + + + + +
Malignant Conversion of Liver Cells apical one. In passage 12 or later, intracellular junctions of close apposition became prominent. The ruthenium red stained layer of the cell surface was similar in thickness and configuration at the different stages of passages (Fig. 8). Carcinomas produced by transplantation in vivo In either s.c. or i.p. transplantation, solid tumors were formed in close association with the surrounding fibrostromal tissues of the recipient animals. As seen in Fig. 9, tumor nodules had the appearance of malignant hepatomas or hepatocellular carcinomas at various degrees of histological differentiation. T u m o r cells were irregularly packed or arranged in interlacing strands, but sometimes showed adenomatous formations. Trabecular patterns were often found with the host fibrostromal network and in association with capillary proliferation. In undifferentiated tumors, individual cells were various in size, and polygonal or occasionally fusiform in shape. Mitotic figures were observed more frequently than in vitro. In thin sections tumor cells appeared related to each other by their parallel, often interdigitating cell surfaces (Fig. 10). In m a n y areas, the interdigitation of cell membranes had the appearance of bile canaliculi. It was noted that tumor cells were often closely apposed with leukocytes and other host cells. Cytochemical tests for ATPase activity gave a positive reaction along the surface membranes of tumor cells (Fig. 11). Only solitary virus particles infrequently were seen within the intercellular spaces. The nuclei sometimes contained intranuclear canaliculi. In the cytoplasm, m a n y peroxisomes [29] were identified by the cytochemical test (Fig. 12). In vitro cultivation of carcinomas Peripheral areas of the tumors produced by the implantation of cultured cells were explanted in the plastic dishes. After 1 day, m a n y round cells migrated out of the explants. This process was followed by the outgrowth of flattened epithelial cells, which appeared to be morphologically identical to those observed in continuous subcultures (Table 2). One million cells were harvested and implanted into newborn rats after 2 and 3 weeks in vitro. In the implanted rats, carcinomas were produced after 2 weeks (Table 1). DISCUSSION
Only a few reports have been published on the development of carcinomas upon in vivo C
531
transplantation of liver cells which were grown in vitro [14]. The present experiment has demonstrated the production in Wistar rats of hepatocellular carcinomas derived from normal adult liver cells in culture. A series of implantation experiments and morphologic examinations have suggested that the cultured cells acquired malignant properties, step by step. This may be true, not only for the growth pattern of cell populations but also for the subcellular differentiation of individual cells in vitro and in vivo. The development of tumors resembling hepatocellular carcinomas in vivo supports the possibility that unspecific epithelial cells observed in vitro were originated from hepatocytes. The findings of peroxisomes and canalicular-like structures in the carcinomas are of particular significance, since these organelles are prominent in differentiated hepatocytes [33]. It seems probable that the induction of a differentiated state occurs in vivo rather than in vitro. In the present investigation, the primary culture was initiated with a highly homogeneous population of hepatocytes which were isolated from normal adult rat liver. Regular examinations during several months in the period of explantation, showed little variation in cell numbers without detectable mitosis. An excessively long period before subcultures would be expected to be selective for a population of cells capable of growth under adverse conditions. It should be pointed out that the culture condition in the present experiment resembles those which Borek [13] found to result in "spontaneous transformation" of established liver epithelial cells. A sublethal damage of cells at the time of liver dissociation or explantation as well as a long residence in quiescent phases might be considered important effects toward "transformation" in vitro. The "transformed" colonies of epithelial cells found in the primary cultures, however, were practically in non-proliferative state. At the first subculture, trypsinization could trigger the conversion of quiescent cells into continuously replicating ones. The mode of cell proliferation changes markedly from that of the parental cells in the course of relatively a few mitotic cycles, and they could soon divide typically every 30 hr or shorter at the logarithmic growth as seen in other established liver cell lines [5, 9, 13, 34]. With repeated passages of the cell line over a period of 2 months, the fast-proliferating cells could produce tumors on in vivo transplantation. The increase of proliferating pool due to an altered pattern
S. Karasaki et al.
532
of cell cycle is a prerequisite for malignant conversion and, in this respect, the sequence of events during early passages in vitro also resembles that observed in premalignant lesions in vivo [22, 24, 35, 36]. T h e present study has demonstrated A and C virus particles in epithelial cells derived from normal rat liver. A close examination of the picture published by Gerschenson et al. [5] also shows a similar particle (but designated as "microbody") in cultured rat liver cells (RLC). Recently, Weinstein et al. [8] have succeeded in demonstrating endogenous A and C particles in 5 hepatoma cultures and in a spontaneously transformed liver cell line, but so far such particles were not detectable in the 5 independently isolated cell cultures from normal rat liver. T h e association of virus particles would not be considered a sign of malignancy, since our cell line rather ceased viral production after passages in vitro when it became tumorigenic. Virus particles of hepatic cells have been found in association with the cell surface and the endoplasmic reticulum or its derivatives [19, 26, 37-39], but little attention has been paid to the presence of virus particles within the nuclear envelope. We found in the primary cultures two types of A particles which appeared to be budding from the nuclear membranes. Coincidentally, there were unusual formations of intranuclear canaliculi which might be derived by deep invaginations of only the inner nuclear m e m b r a n e [40]. T h e developm e n t of intranuclear membranes in nondividing cells is suggestive of a continued synthesis of the inner nuclear membrane under an interaction of viral ligaments at a period when nucleoplasm volume is stable. This type of intranuclear membranes were also demonstrated in various neoplastic cells which could produce A or C type virus particles [12, 37, 38, 41]. An interaction of viral ligaments might cause abnormal membrane formations. W h e n released from a mitotic block by subculture, the proliferating cells had transitory cytoplasmic structures, such as annulate lamellae and paired cisternae. These organelles have been suggested to arise from the nuclear envelope [42-44]. Their apparent structural relationship to endoplasmic reticulum has been also indicated in actively growing or proliferating cells in normal and malignant tissues [25,
38, 39, 42-44]. T h e emergence of these transitory organelles in the present cell line might result from viral interaction with the nuclear envelope, since it is frequently accompanied by virus particles in the endoplasmic vesicles. T h e sequential phenotypic expressions at the specific stages in vitro suggest a developmental role of oncorna virus in subcellular differentiation leading to a neoplastic state. An essential prerequisite for the malignant conversion in vitro would be alterations which eventually influence the cell surface properties characteristic of neoplastic cells [ 13, 21, 23, 24, 30, 39, 45]. With the Wachstein-Meisel method for cytochemical demonstration of ATPase activity, a positive reaction at the surface m e m b r a n e of cells was found concomitant with the acquisition of tumorigenicity in the cell population. An intense ATPase reaction was biochemically demonstrated at the surface of intact ceils from all tumorigenic liver cell lines tested, including the present line [46]. Similar surface changes in phosphatase localizations have been reported in hepatocellular carcinomas during in vivo transplantation [39, 44] and in liver parenchyma during the administration of a carcinogenic azo dye [23, 24]. Since there was a close temporal correlation between the cytochemical pattern in vitro (Table 2) and tumorigenicity in vivo (Table 1), the cell surface alteration would be considered as a useful marker for a further study of malignant conversion of epithelial cells. It was recognized during passages in vitro that epithelial cells decreased their adhesiveness toward the plastic substratum while intercellular junctions of close apposition became gradually prominent. T h e implantation of cultured ceils into rats exhibited histological differentiation which might involve the process of preferential convergence of cells within the stromal tissue of a recipient. The differential adhesiveness as well as the increase of surface ATPase activity of the epithelial cells would facilitate for a growth advantage upon in vivo transplantation.
Acknowledgement--We
t h a n k L u c i a n o Borsato for v a l u a b l e t e c h n i c a l help, a n d B e r n a r d Szirth for assistance in p h o t o g r a p h y . W e also t h a n k A n d r d e L e M y r e , A l i n e F a r n e r a n d Alec T r e t i a k w h o p r e p a r e d cell cultures d u r i n g the course of this work.
REFERENCES
1.
R. W. ALEXANDERand J. W. GRISHAM,Explant culture of rat liver. I. Method, morphology, and cytogenesis. Lab. Invest. 22, 50 (1970).
Malignant Conversion o f Liver Cells
2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12. 13.
14. 15.
16.
17. 18.
19.
20.
21. 22. 23. 24.
D . M . BISSELL,L. E. HAMMAKERand U. A. MEYER, Parenchymal cells from adult rat liver in nonproliferating monolayer culture. I. Functional studies. J. Cell Biol. 59, 722 (1973). M . N . BERRY and D. S. FRm~-D, High-yield preparation of isolated rat liver parenchymal cells. A biochemical and fine structural study. J. Cell Biol. 4~, 506 (1969). G. D~ LAMmArCD~ and A. SIMAaD, l~tude biochimique d'h6patocytes en culture provenant de rats normaux adultes. Rev. canad. Biol. 34, 111 (1975). L . E . GERSCHENSON, T. OKIGAKI, M. ANDERSSON, J. MOLSON and M. E. DAVIDSON, Fine structural and growth characteristics of cultured rat liver cells. Expt. Cell Res. 71, 49 (1972). P . T . I'ZI,E, Cultures from adult rat liver cells. I. Establishment of monolayer cell-cultures from normal liver. J. Cell Physiol. 78, 281 (1971). T. OKIGAKIand S. O. O~UAN,Morphological inconsistency in liver cells in culture. Proc. Japan Acad. 50, 517 (1974). I . B . WEINSTEIN,J. M. OR~.NSTEIN, R. GEBErtT, M. E. KAIOH~ and U. C. STADL~S,Growth and structural properties of epithelial cell culture established from normal rat liver and chemically induced hepatomas. Cancer Res. 35, 253 (1975). G . M . WILLIAMSand J. M. GuNN, Long-term cell culture of adult rat liver epithelial cells. Exp. Cell Res. 89, 139 (1974). G . M . WILLIAMS,K. STROM-BEROand R. KROES, Cytoehemical and ultrastructural alterations associated with confluent growth in cell cultures of epithelial-like cells from rat liver. Lab. Invest. 29, 293 (1973). G . M . WILLIAMS,E. K. WEISBURGERand J. H. WEISBURGER,Isolation and long-term culture of epithelial-like cells from rat liver. Exp. Cell Res. 69, 106 (1971). G. YAsUZUMIand R. SUOIHARA,The fine structure of nuclei as revealed by electron microscopy. The fine structure of Ehrlich ascites tumor cell nuclei in preprophase. Exp. Cell Res. 37, 207 (1965). C. BOg~K, Neoplastic transformation in vitro of a clone of adult liver epithelial cells into differentiated hepatoma-like cells under conditions of nutritional stress. Proc. nat. Acad. Sci. (Wash.), 69, 956 (1972). Y. OSHIRO, L. E. GERSCHENSONand J. A. DIPAoLO, Carcinomas from rat liver cells transformed spontaneously in culture. Cancer Res. 32, 877 (1972). J. SATO, M. NAMBA,K. UsuI and D. NAOANO, Carcinogenesis in tissue culture. VIII. Spontaneous malignant transformation of rat liver cells in long-term culture. Jap. d. exp. Med. 38, 105 (1968). H. KATSUTAand T. TAKAOKA,Carcinogenesis in tissue culture. XIV. Malignant transformation of rat liver parenchymal cells treated with 4-nitroquinoline 1-oxide in tissue culture, d. nat. Cancer Inst. 49, 1563 (1972). K. KOSHIBA,M. NAMBAand T. ODA, Electron microscopic studies on cultured rat liver cells transformed by 4-nitroquinoline 1-oxide. Gann 61, 233 (1970). J. SATO,Malignant transformation in culture of rat liver cells treated with or without 3'-methyl-4-dimethylaminoazobenzene. In Cancer Cells in Culture. (Edited by H. KATSUTA) p. 335. University of Tokyo Press, Tokyo (1968). G . M . WILLIAMS,J. M. ELLIOTTandJ. H. WEISBUROER,Carcinoma after malignant conversion in vitro of epithelial-like cells from rat liver following exposure to chemical carcinogens. Cancer Res. 33, 606 (1970). C. ALTANER and E. HLAVAYOVA,Transformation of rat liver cells with chicken sarcoma virus B77 and murine sarcoma virus, d. Virol. 11, 177 (1973). K . K . SANFORD, Biologic manifestation of oncogenesis in vitro: A critique. J. nat. Cancer Inst. 53, 1481 (1974). S. KARASAKI,The fine structure of proliferating cells in preneoplastic rat livers during azo-dye carcinogenesis. J. Cell Biol. 40, 322 (1969). S. KA~ASAKI, Subcellular localization of surface adenosine triphosphatase activity in preneoplastic liver parenchyma. Cancer Res. 32, 1703 (1972). S. KARASAKI,Cell proliferation and subcellular localization of alkaline phosphatase activity in rat liver during azo dye carcinogenesis. Cancer Res. 35, 482 (1975).
533
S. Karasaki et al.
534
25.
26. 27.
28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46.
L.P. MERKOW,S. M. EPSTEIN, E. FARBER,M. PARDOand B. BARTAS,Cellular analysis of liver carcinogenesis. III. Comparison of the ultrastructure of hyperplastic liver nodules and hepatocellular carcinomas induced in rat liver by 2-fluorenyl acetamide. J. nat. Cancer Inst. 43, 33 (1969). I.B. WEINSTEIN, R. GEBERT, V. C. STADLER,J. M. ORENSTEINand R. AXLE, Type C virus from cell cultures of chemically induced rat hepatomas. Science 178, 1098 (1972). A . B . NOVlKOFF and S. GOLDFISHER,Visualization of peroxisomes (microbodies) and mitochondria with diaminobenzidine. J. Histochem. Cytochem. 17, 675 (1969). M. WACHSTEIN and H. MEISEL, Histochemistry of hepatic phosphatase at physiologic pH. Amer. J. din. Pathol. 27, 13 (1957). Z. HRUBAN, E. L. VIGIL, A. SLEVERS and E. HOPKINS, Microbodies--constituent organelles of animal cells. Lab. Invest. 27, 184 (1972). A. MARTINEZ-PALOMO,C. BRAISLOVSKYand W. BERNHARD, Ultrastructural modifications of the cell surface and intercellular contacts. Cancer Res. 29, 925 (1969). W. BERNHARD, The detection and study of tumor viruses with the electron microscope. Cancer Res. 20, 712 (1960). D . G . CHASEand L. P. PII~O, Expression of A- and C-type particles in early mouse embryo. J. nat. Cancer Inst. 51, 1971 (1972). C. ROUILLER and A. M. JEZEQUIEL, Electron microscopy of the liver. In The Liver. Morphology, Biochemistry, Physiology. I. (Edited by C. ROUILLER) p. 195. Academic Press, New York (1963). E.B. THOMPSON, G. M. TOMKINSand J. F. CURRAN, Induction of tyrosine-aketo glutarate transaminase by steroid hormones in a newly established tissue culture line. Proc. nat. Acad. Sci. (Wash.) 56, 296 (1966). W. OEnLERT, Cellular proliferation in carcinogenesis. Cell Tissue Kinet. 6, 325 (1973). A. SIMARD,G. COUSINEAUand R. DAOUST,Variations in the cell cycle during azo dye carcinogenesis. J. nat. Cancer Inst. 41, 1257 (1968). S. KARASAKI, Virus particles associated with the transplantable Novikoff hepatoma in the rat. Cancer Res. 29, 1313 (1969). J. LOCKER, P. J. GOLDBLATTand J. LEIGHTON,Some ultrastructural features of Yoshida ascites hepatoma. Cancer Res. 28, 2039 (1968). A . B . NOVIKOFF and L. BIEMPICE, Cytoehemical and electron microscopic examination of Morris 5123 and Reuber H-35 hepatomas after several years of transplantation. Gann Monogr. 1, 65 (1966). S. KARASAKI, An electron microscope study of intranuclear canaliculi in Novikoff hepatoma cells. Cancer Res. 30, 1736 (1970). E. BUCCIARELLI, Intranuclear cisternae resembling structures of the Golgi complex. J. Cell Biol. 30, 664 (1966). H. HANAOKA and B. FRIEDMAN, Paired cisternae in human tumor cells. J. Ultrastruct. Res. 32, 323 (1970). R . O . KELLY,Ultrastructural comparisons of paired cisternae in leukemic and mesenchymal cells. Anat. Rec. 171, 559 (1971). R . a . KESSEL,Annulate lamellae. J. Ultrastruct. Res. Suppl. 10, 1 (1968). E. ESSNER and M. D. REUBER, Localization of acid phosphatase activity in well-differentiated hepatocellular carcinoma 146. J. nat. Cancer Inst. 479 25 (1971). S. KARASAKI, Surface membrane ATPase activity in transformed rat liver epithelial cell lines. J. Cell Biol. 679 200a (1975).
Ultrastructural Analysis of Malignant Conversion In Vitro of Epithelial Cells Derived from Normal Adult Rat Liver* S. KARASAKI~$, A. SIMARDJ" and G. DE LAMIRANDE§ $ Institut du Cancer de Montrdal, Centre Hospitalier Notre-Dame, and ~Ddpartement d'Anatomie et §Ddpartement de Biochimie, Universitd de Montrdal, Montrgal, Canada Abstract A tumorigenic cell line of epithelial-type was establishedfrom a long-term culture of isolated liver cells from a normal adult Wistar rat. Electron microscopic examinations of foci of transformed epithelial cells in the primary culture revealed production of virus particles in association with membranes of the nuclear envelope, endoplasmic reticulum and cell surface. The epithelial cells were found in quiescent state and had intranuclear canalicular membranes. By trypsinization, the cells were initiated into a rapid replication and showed subcellular differentiation. Annulate lamellae and paired cisternae emerged in passage 1. Viral production ceased at this stage. Close apposition of adjacent cells became prominent in passage 9, when the cell line first revealed tumorigenicity upon transplantation into neonatal Wistar rats. Concomitantly, cytochemical tests gave a positive reaction of A TPase activity on the plasma membranes. Surface A TPase activity continued to show in the epithelial cells following passages in vitro as well as in tumor cells in vivo. The possible origin of epithelial cells in vitro from hepatocytes was suggested by the demonstration of peroxisomes and bile canalicular-like structures in the carcinoma cells.
of liver cells [8, 17, 26]. O f these reports, Weinstein et al. [8, 26] have focused on the presence of oncorna virus particles in rat liver cell lines and their correlation with transformed cells. The sequential process of in vitro neoplastic transformation is very little understood at the subcellular level. This paper reports the establishment and morphological characterization of a tumorigenic cell line of epithelial-type, derived from the liver of a normal adult rat. The studies of cells in the primary culture and subcultures involve fine structural and cytochemical observations with the objective of obtaining clues to the nature and sequence ofsubcellular events which may constitute cytodifferentiation toward malignant conversion in vitro. The carcinomas, produced by the implantation of cultured cells in rats, are ultrastructurally examined in special attention to cytochemical properties of differentiated hepatocytes.
INTRODUCTION ISOLATED hepatocytes from normal rat liver usually have a limited life span in vitro [1-4]. Under certain conditions, however, liver cells m a y continue to multiply [5-12] and eventually become malignant during years of long term cultivation [13-15]. Malignant conversion in vitro of cultured liver cells has been induced or accelerated by treatments with chemical carcinogens [8, 16-19] or oncorna viruses [20]. The detection of malignant conversion in vitro of epithelial cells is particularly difficult, since little alteration is induced in cell morphology at the critical step [21]. Although m a n y studies were performed on hepatocarcinogenesis in vivo [22-25], there are few reports of subcellular analyses on in vitro transformation
Accepted 26 January 1976 *This work was supported by grants from the National Cancer Institute of Canada, le Minist~re des Affaires sociales du Q.udbec, La Fondation J. H. Biermans and Les Fondations J. Rhdaume. ++Research Associate of the National Cancer Institute of Canada.
MATERIAL AND METHODS Cell separation Rats of the Wistar strain were obtained from 527
528
S. Karasaki et al.
Canadian Breeders, Inc., St. Constant, Que., Canada. The cell line studied here was derived from the liver of a normal male weighing 200 g. The liver was dissociated on M a y 10, 1973. The cell separation technique employed was essentially the same as that described by Berry and Friend [3]. After perfusion through the hepatic vein with Ca2+-deficient Hank's solution containing 0.05% hyaluronidase (Sigma) and 0.025% collagenase II (Sigma), liver ceils were suspended in a mixture (1:1) of Ham's F12 culture medium (GIBCO) and of Hank's solution containing 0-5 M sucrose and passed through two successive sieves with 250 and 40/l mesh. The enzymatic dissociation of an adult rat liver yielded 2"5 x 10 s cells. The cell population consisted almost exclusively of hepatocytes (99.1%) as determined with Carnoy-fixed smears stained with hematoxylin-eosin. Cell culture
The standard culture medium was H a m ' s F12 containing 100 units of penicillin G and 100 pg of streptomycin per ml and, supplemented with 5 or 10°/O fetal calf serum (GIBCO). Isolated hepatocytes were plated at 37°C in plastic T-flasks ( 2 5 c m 2, Falcon). Cellular concentration was 5 x l 0 6 per 10 ml. Within 6 hr of explanting in a plastic flask, a majority of spherical hepatocytes were packed in a layer of aggregates. They had an angular outline and were loosely attached to the plastic surface during the 1st week period. The medium was partially changed 2 or 3 times a week and were maintained in the same flasks from 4 months to more than 18 months in some cases. Regular determination of cell numbers in flasks showed little variation or a very slow decrease. Six months after explantation, 7 separate cultures showed signs of cell growth. O n J a n u a r y 4, 1974, the first subculture was performed on one of the flasks. To dislodge the cells, a mechanical dispersion method was applied after 10rain of treatment with a 0.25% trypsin solution at 37°C. Further passages in vitro were continuously done at 5-8 days intervals with trypsinization. Plating efficiency in earlier passages was less than 5% of the cells. In passage 4, cells grew well in monolayer (Fig. 1). In passage 6 or later, the cells were easily detachable by trypsinization. In passage 12, the logarithmic growth was readily resumed after a lag period of 18 to 24 hr. At this point, the plating efficiency was in excess of 20% and the population doubling time was less than 30 hr in the
4,
°-4
/,,-'"
-12-
Fig. I. Growth patterns of cultured liver cells at the 4th ( - - O - -) and 12 th ( - - 0 - - ) passages. Cells were placed in a series of T-5 flasks (75 cm 2) to each of which 15 ml of Ham's F12 mediumplus 10% fetal bovine serum was added. Each day, the culture was trypsinized and the number of cells was measured in Coulter counter.
logarithmic phase (Fig. 1). The saturation of cell density resulted in the inhibition of growth (Fig. 5). More than 90 passages have been done over a period of 20 months. Tumorigenicity
Newborn Wistar rats (6-18 hr after birth) were used for tumorigenicity tests of the cultured cells. Cells were dissociated by either trypsinization or mechanical dispersion. A suspension o f l 0 6 to 2 x 106 cells in 0.05-0.1 ml of culture medium was injected subcutaneously or intraperitoneally. When transplanted either i.p. or s.c. into newborn rats with 2 x 106 cells from passage 6, no tumors were formed (Table 1). In the same batch of animals, positive results were obtained on s.c. transplantation of an established cell line, R L C [4, 27]. 2 x 106 cells of passage 9 could produce tumors on i.p. transplantation. Further passages developed tumors on s.c. transplantation of 1-2 x 106 cells with a latency period of 10-18 days (Table 1). Some of the tumors were explanted and grown in vitro. A s.c. inoculation of 106 cells gave a solid tumor of 10 cm 3 volume after 4 weeks and killed the recipient. I.p. transplants developed into many solid tumors on the surface of peritoneum and the diaphragm. Metastasis into the eye was found in an i.p. transplant. Routine histologic examinations were done on Carnoy-fixed, paraffin-embedded and toluidine blue-stained tissues. Electron microscopy
The cells in plastic culture flasks were fixed in situ for 30 min to 1 hr with 2.5% glutar-
Fig. 11. A TPase reaction in glutaraldehyde-fixed tumor section. A positive reaction on the pattern of lead phosphate precipitates follows the course of the plasma membranes (arrows). Intranuclear canaliculi ( NC). x 12,000. Fig. 12. Diaminobenzidine-H20 2 reaction in glutaraldehyde-fixed tumor section. Peroxidase activity is seen located in microbodies of carcinoma cells (arrow). x 30,000.
Fig. 9. Photomicrograph of a solid tumor produced by i.p. transplantation of the cultured cells in passage 9. The peripheral portion of a solid tumor shows three parts with different histological patterns as well as variation in staining characteristics. Fibro-stromal tissues (S) and capillaries (E) of the host. Carnoy-toluidine blue. x 225. Fig. I O. Electron micrograph of a part of carcinoma tissue. Bile canalicular-like surfaces (arrows). Opaque amorphous material resembling biliary products, x 6,750.
Fig. 7. A TPase reaction in epithelial cells from a preconfluent culture of passage 9. The surface membranes of adjacent cells were seen covered with bands of opaque reaction product. Regional differences in the surface deposits of the reaction product, ranging from few tiny particles to continuous bands, x 7,500. Fig. 8. Ruthenium red staining of epithelial cells from a confluent of passage 20. In tangential section of 3 adjacent cells, the surface membranes are closely apposed and covered with an electron opaque layer of ruthenium red stained coat. Pinocytotic vesicles (arrows). x 15,000.
Fig. 6. (a)-(d).
Rat liver epithelial cells in passage 1.
(a) Paired cisternae (PC) in the cytoplasm. Small A particle within a smooth vesicle (a). Intranuclear canaliculi (arrows). A large nucleolus with prominent granular components. Uniformly distributed chromatin, x 15,000. (b) Perpendicular section of annuli in cytoplasmic annulate lamellae ( AL). Small A particle within a smooth vesicle (a). x 52,500. (c) Tangential section of annuli in cytoplasmic annulate lamellae ( AL). Parallel arrays of rough endoplasmic reticulum cisternae. Large A particles within a smooth vesicle (A). x 30,000. (d) Cytoplasmic annulate lamellae (AL) in continuity with rough endoplasmic reticulum. Large A particle within a smooth vesicle (A). x 22,500.
Fig. 4. Liver epithelial cells in the primary culture (after 7 months). Intercellular and intracisternal virus particles. Budding (1), immature (2) and mature (3)forms of Cparticles. Large A particles in the cisternae of nuclear envelope. The nucleolus (NO) show a decreased amount of granular components which are segregated from fibrillar components, x 30,000. Fig. 5. Liver epithelial cells in the primary culture (after 7 months). Free and budding form~ of intracisternal A particles. Small A particles (a) within the perinuclear eisternae and the vesicles of endoplasmic reticulum. Large A particles (A) within cytoplasmic vacuoles. Intranuclear membranes (arrow) and lipid (L). x 67,500.
Fig. 2. Electron micrographs of rat liver epithelial cells in the primary culture (after 7 months). Intranuclear canaliculi (IN). Virus particles (arrows). Undulated cell surface, x 12,000. Fig. 3. Electron micrographs of rat liver epithelial cells in the primary culture (after 7 months). Large and small nuclei with different patterns of chromatin distribution. Virus particles (arrows). Endoplasmic reticulum with flattened sacs, the content of which are moderately dense, x 22,500. (to face page 528)
Malignant Conoersion: o f L'iver Cells Table 1.
Tumorigenicity of cultured epithelioid cellsfrom rat liver
Days after subculture*
Passage number]"
44 55 65 80 98
--6---9---12-14 --9--C1--1--
118
157 389
529
--20--
--16--C1--2---50--
Tumor incidence + i.p. § s.c. § 0/3 2/3 -2/2 -2/4 ---
0/2 0/4 1/3 2/5 5/6 3/4 2/2 3/4
Average latency period (days) --15 18 12 13 14 10
*Plus 237 days in the primary culture of isolated hepatocytes. ].Passage number in vitro, except C1 (1 passage in vivo). ++Number of animals with carcinoma/Number of newborns inoculated. §Route of implantation of 1-2 x 106 cells per animal. aldehyde (Ladd) in 0.1 M sodium cacodylateHC1 buffer (pH 7.1), washed for 1-4 hr in 7"5% sucrose solution, post-fixed for 1 hr with 1.4% OsO4 in 0.1 Ms-collidine buffer (pH 7.1), dehydrated in a graded ethanol-water series and embedded in Epon. Sections were prepared for both light and electron microscopy. Thin sections were stained with uranyl acetate and lead citrate, and were viewed in a J E M - 7 A electron microscope. Specimens of tumor tissues were similarly processed.
Cytochemistry To detect cytochemical alterations of surface membrane of the cells during passages in vitro and in vivo, the method of Wachstein and Meisel [28] for ATPase demonstration was applied on glutaraldehyde-fixed cells in situ in plastic flasks or on sections of fixed tumors. The method of Novikoff and Goldfisher [27] was similarly applied for detection of peroxisomes [29]. Fixatives containing ruthenium red were also used to identify carbohydratecontaining structures on the cell surface [30]. RESULTS
The primary culture During 6 months period the originally explanted hepatocytes remained attached over the surface of flask bottom. Each of 7 separate cultures from the single preparation ( < 2"5 x 10 a cells) contained 3-10 foci of colonies of epithelial cells and sparsely scattered stellatetype cells. The foci of epithelial cells grew very slowly, although mitoses were never detected. The ultrastructural examinations were performed on the colonies of epithelial cells after
7 and 8 months in culture. It appeared that they had lost almost all of their original tissue characteristics at the subceUular level, but they were found to vary markedly from one another in fine structure. The cross section of epithelial cells in situ showed contacts of the plasma membrane to the plastic substratum and an accumulation of microfilaments and microtubules adjacent to the membrane, suggesting a strong adhesion of cells with the substratum. Cell-to-cell contacts were mostly focal at the crests of surface undulation (Fig. 2). Cells contained more than one nucleus, which demonstrated different patterns of chromatin distribution (Fig. 3). The nuclei were extremely deformed and lobulated in outline, frequently possessing deep cytoplasmic invaginations (Fig. 2-5). The nucleoplasm included canalicular membranes, of which structure appeared to be similar to that of the inner nuclear membrane (Fig. 2). In the cytoplasm, there were membranous profiles of endoplasmic reticulum scattered singly or in parallel arrays (Fig. 2-5). Numerous structures resembling oncorna virus particles were frequently seen (Fig. 2-5). Extracellular C particles [31] and two types ofintracisternal A particles [32] were identified (Fig. 4 and 5). C particles measured 90-100 nm dia and were formed by a typical process of budding from the cell membrane into the intercellular space (Fig. 4). Immature C particles comprised a relatively electronlucent core surrounded by an electron dense shell (Fig. 4). Mature C particles contained a central dense core surrounded by an irregularly contoured membrane (Fig. 4). Small and large types of A particles were present in the cisternae of nuclear envelope and the vesicles of endoplasmic reticulum (Fig. 5). Particle size ranged from 70-80 nm for the small type and
530
S. Karasaki et al.
from 9 0 - 1 1 0 n m for the large type (Fig. 5). Both types of particles appeared to contain 2 concentric dense shells and an electron lucent center (Fig. 5). Budding forms of both types of particles were seen from the m e m b r a n e s of either endoplasmic reticulum or nuclear envelope (Fig. 5). Subcultures T h e cultures of passage 1 contained epithelial cells mixed with stellate-type cells. Mitoses were frequently seen. Following further subcultures, the cells became more homogeneous in size and shape. Comparative studies at the ultrastructural level were performed and the subcellular features in epithelial cells in the p r i m a r y culture as well as in subcultures are summarized in Table 2 with reference to the sequence of passage numbers. In passage 1, the majority of cells showed less irregularities of nuclear fine structure when compared to those of p r i m a r y culture. O n l y a few exhibited intranuclear canalicular membranes (Fig. 6a). Paired cisternae (Fig. 6a) and annulate lamellae (Fig. 6b-d) were frequently found in passage 1. These cytoplasmic m e m b r a n o u s structures morphologically resembling the nuclear membrane were often located adjacent to the nucleus (Fig. 6a). T h e y were also found in continuity with the m e m b r a n e s of endoplasmic reticulum (Fig. 6d). Virus particles were seen within the vesicles of endoplasmic reticulum adjacent to annulate lamellae or paired cisternae (Fig. 6a-d) and rarely in association Table 2.
with the cell surface, but none within the perinuclear cisternae. T h e fine structure of nuclei and cytoplasm did not appear to change remarkably with passage number. There were few specializations of cytoplasmic organelles, accompanying the confluency of cells. R o u g h endoplasmic reticulum cisternae were quite p r o m i n e n t in post-confluent cultures. Peroxisomes and glycogen particles were not detectable by cytochemical methods. Microfilaments adjacent to the plasma membranes were rare at the preconfluency and increased significantly at the confluency. A few virus particles were detectable by passage 6, while later their detection was seldom. In passages 4 and 6, the junctions of close apposition between individual cells were mostly focal and the intracellular dilations were evident. Using the Wachstein-Meisel technique, ATPase reaction was not seen by passage 8 (Table 2). In passage 9, however, some colonies showed the reaction product at the outer surface of plasma membranes of epithelial cells (Fig. 7). T h e distribution of the reaction product varied a m o n g individual cells, such as complete absence from some and its restriction to certain regions on the surface of others (Fig. 7). T h e ATPase-positive cells were ultrastructurally indistinguishable from neighboring cells which showed no activity. All epithelial cells from passage 12 exhibited reaction products at r a n d o m along the entire plasma membranes, although it was more a b u n d a n t on the apposed surfaces t h a n the
Sequentialalterations of cultured epithelial cells after in vitro and in vivo passages Cytochemical reaction]"
Electron microscopic detection]" Passage number*
Virus particles
Intranuclear canalicular membranes
--0---1---4--
+ +/-/+
+ -/+ -/+
--6--
--/+
--8.
.
. 112 . --14 . --9--C1--1---20 . - - 16--C1--2 . --50 .
.
--9
.
.
. . .
. . -/+
Surface close appositions
Surface ATPase activity
-/+ -/+ -/+
----
--/+
--
/+
-/+
. .
.
--
.
-I+ .
. .
-/+ + -/+
--
.
. . .
Cytoplasmic annulate lamellae
. .
*Passage number in vitro, except C1 (1 passage in vivo). I"+ : Frequent detection in thin sections of ceils. +/-- : infrequent, - / + : rare, --: seldom or never encountered.
+ + + + + + +/-
+/+ + + + + +
Malignant Conversion of Liver Cells apical one. In passage 12 or later, intracellular junctions of close apposition became prominent. The ruthenium red stained layer of the cell surface was similar in thickness and configuration at the different stages of passages (Fig. 8). Carcinomas produced by transplantation in vivo In either s.c. or i.p. transplantation, solid tumors were formed in close association with the surrounding fibrostromal tissues of the recipient animals. As seen in Fig. 9, tumor nodules had the appearance of malignant hepatomas or hepatocellular carcinomas at various degrees of histological differentiation. T u m o r cells were irregularly packed or arranged in interlacing strands, but sometimes showed adenomatous formations. Trabecular patterns were often found with the host fibrostromal network and in association with capillary proliferation. In undifferentiated tumors, individual cells were various in size, and polygonal or occasionally fusiform in shape. Mitotic figures were observed more frequently than in vitro. In thin sections tumor cells appeared related to each other by their parallel, often interdigitating cell surfaces (Fig. 10). In m a n y areas, the interdigitation of cell membranes had the appearance of bile canaliculi. It was noted that tumor cells were often closely apposed with leukocytes and other host cells. Cytochemical tests for ATPase activity gave a positive reaction along the surface membranes of tumor cells (Fig. 11). Only solitary virus particles infrequently were seen within the intercellular spaces. The nuclei sometimes contained intranuclear canaliculi. In the cytoplasm, m a n y peroxisomes [29] were identified by the cytochemical test (Fig. 12). In vitro cultivation of carcinomas Peripheral areas of the tumors produced by the implantation of cultured cells were explanted in the plastic dishes. After 1 day, m a n y round cells migrated out of the explants. This process was followed by the outgrowth of flattened epithelial cells, which appeared to be morphologically identical to those observed in continuous subcultures (Table 2). One million cells were harvested and implanted into newborn rats after 2 and 3 weeks in vitro. In the implanted rats, carcinomas were produced after 2 weeks (Table 1). DISCUSSION
Only a few reports have been published on the development of carcinomas upon in vivo C
531
transplantation of liver cells which were grown in vitro [14]. The present experiment has demonstrated the production in Wistar rats of hepatocellular carcinomas derived from normal adult liver cells in culture. A series of implantation experiments and morphologic examinations have suggested that the cultured cells acquired malignant properties, step by step. This may be true, not only for the growth pattern of cell populations but also for the subcellular differentiation of individual cells in vitro and in vivo. The development of tumors resembling hepatocellular carcinomas in vivo supports the possibility that unspecific epithelial cells observed in vitro were originated from hepatocytes. The findings of peroxisomes and canalicular-like structures in the carcinomas are of particular significance, since these organelles are prominent in differentiated hepatocytes [33]. It seems probable that the induction of a differentiated state occurs in vivo rather than in vitro. In the present investigation, the primary culture was initiated with a highly homogeneous population of hepatocytes which were isolated from normal adult rat liver. Regular examinations during several months in the period of explantation, showed little variation in cell numbers without detectable mitosis. An excessively long period before subcultures would be expected to be selective for a population of cells capable of growth under adverse conditions. It should be pointed out that the culture condition in the present experiment resembles those which Borek [13] found to result in "spontaneous transformation" of established liver epithelial cells. A sublethal damage of cells at the time of liver dissociation or explantation as well as a long residence in quiescent phases might be considered important effects toward "transformation" in vitro. The "transformed" colonies of epithelial cells found in the primary cultures, however, were practically in non-proliferative state. At the first subculture, trypsinization could trigger the conversion of quiescent cells into continuously replicating ones. The mode of cell proliferation changes markedly from that of the parental cells in the course of relatively a few mitotic cycles, and they could soon divide typically every 30 hr or shorter at the logarithmic growth as seen in other established liver cell lines [5, 9, 13, 34]. With repeated passages of the cell line over a period of 2 months, the fast-proliferating cells could produce tumors on in vivo transplantation. The increase of proliferating pool due to an altered pattern
S. Karasaki et al.
532
of cell cycle is a prerequisite for malignant conversion and, in this respect, the sequence of events during early passages in vitro also resembles that observed in premalignant lesions in vivo [22, 24, 35, 36]. T h e present study has demonstrated A and C virus particles in epithelial cells derived from normal rat liver. A close examination of the picture published by Gerschenson et al. [5] also shows a similar particle (but designated as "microbody") in cultured rat liver cells (RLC). Recently, Weinstein et al. [8] have succeeded in demonstrating endogenous A and C particles in 5 hepatoma cultures and in a spontaneously transformed liver cell line, but so far such particles were not detectable in the 5 independently isolated cell cultures from normal rat liver. T h e association of virus particles would not be considered a sign of malignancy, since our cell line rather ceased viral production after passages in vitro when it became tumorigenic. Virus particles of hepatic cells have been found in association with the cell surface and the endoplasmic reticulum or its derivatives [19, 26, 37-39], but little attention has been paid to the presence of virus particles within the nuclear envelope. We found in the primary cultures two types of A particles which appeared to be budding from the nuclear membranes. Coincidentally, there were unusual formations of intranuclear canaliculi which might be derived by deep invaginations of only the inner nuclear m e m b r a n e [40]. T h e developm e n t of intranuclear membranes in nondividing cells is suggestive of a continued synthesis of the inner nuclear membrane under an interaction of viral ligaments at a period when nucleoplasm volume is stable. This type of intranuclear membranes were also demonstrated in various neoplastic cells which could produce A or C type virus particles [12, 37, 38, 41]. An interaction of viral ligaments might cause abnormal membrane formations. W h e n released from a mitotic block by subculture, the proliferating cells had transitory cytoplasmic structures, such as annulate lamellae and paired cisternae. These organelles have been suggested to arise from the nuclear envelope [42-44]. Their apparent structural relationship to endoplasmic reticulum has been also indicated in actively growing or proliferating cells in normal and malignant tissues [25,
38, 39, 42-44]. T h e emergence of these transitory organelles in the present cell line might result from viral interaction with the nuclear envelope, since it is frequently accompanied by virus particles in the endoplasmic vesicles. T h e sequential phenotypic expressions at the specific stages in vitro suggest a developmental role of oncorna virus in subcellular differentiation leading to a neoplastic state. An essential prerequisite for the malignant conversion in vitro would be alterations which eventually influence the cell surface properties characteristic of neoplastic cells [ 13, 21, 23, 24, 30, 39, 45]. With the Wachstein-Meisel method for cytochemical demonstration of ATPase activity, a positive reaction at the surface m e m b r a n e of cells was found concomitant with the acquisition of tumorigenicity in the cell population. An intense ATPase reaction was biochemically demonstrated at the surface of intact ceils from all tumorigenic liver cell lines tested, including the present line [46]. Similar surface changes in phosphatase localizations have been reported in hepatocellular carcinomas during in vivo transplantation [39, 44] and in liver parenchyma during the administration of a carcinogenic azo dye [23, 24]. Since there was a close temporal correlation between the cytochemical pattern in vitro (Table 2) and tumorigenicity in vivo (Table 1), the cell surface alteration would be considered as a useful marker for a further study of malignant conversion of epithelial cells. It was recognized during passages in vitro that epithelial cells decreased their adhesiveness toward the plastic substratum while intercellular junctions of close apposition became gradually prominent. T h e implantation of cultured ceils into rats exhibited histological differentiation which might involve the process of preferential convergence of cells within the stromal tissue of a recipient. The differential adhesiveness as well as the increase of surface ATPase activity of the epithelial cells would facilitate for a growth advantage upon in vivo transplantation.
Acknowledgement--We
t h a n k L u c i a n o Borsato for v a l u a b l e t e c h n i c a l help, a n d B e r n a r d Szirth for assistance in p h o t o g r a p h y . W e also t h a n k A n d r d e L e M y r e , A l i n e F a r n e r a n d Alec T r e t i a k w h o p r e p a r e d cell cultures d u r i n g the course of this work.
REFERENCES
1.
R. W. ALEXANDERand J. W. GRISHAM,Explant culture of rat liver. I. Method, morphology, and cytogenesis. Lab. Invest. 22, 50 (1970).
Malignant Conversion o f Liver Cells
2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12. 13.
14. 15.
16.
17. 18.
19.
20.
21. 22. 23. 24.
D . M . BISSELL,L. E. HAMMAKERand U. A. MEYER, Parenchymal cells from adult rat liver in nonproliferating monolayer culture. I. Functional studies. J. Cell Biol. 59, 722 (1973). M . N . BERRY and D. S. FRm~-D, High-yield preparation of isolated rat liver parenchymal cells. A biochemical and fine structural study. J. Cell Biol. 4~, 506 (1969). G. D~ LAMmArCD~ and A. SIMAaD, l~tude biochimique d'h6patocytes en culture provenant de rats normaux adultes. Rev. canad. Biol. 34, 111 (1975). L . E . GERSCHENSON, T. OKIGAKI, M. ANDERSSON, J. MOLSON and M. E. DAVIDSON, Fine structural and growth characteristics of cultured rat liver cells. Expt. Cell Res. 71, 49 (1972). P . T . I'ZI,E, Cultures from adult rat liver cells. I. Establishment of monolayer cell-cultures from normal liver. J. Cell Physiol. 78, 281 (1971). T. OKIGAKIand S. O. O~UAN,Morphological inconsistency in liver cells in culture. Proc. Japan Acad. 50, 517 (1974). I . B . WEINSTEIN,J. M. OR~.NSTEIN, R. GEBErtT, M. E. KAIOH~ and U. C. STADL~S,Growth and structural properties of epithelial cell culture established from normal rat liver and chemically induced hepatomas. Cancer Res. 35, 253 (1975). G . M . WILLIAMSand J. M. GuNN, Long-term cell culture of adult rat liver epithelial cells. Exp. Cell Res. 89, 139 (1974). G . M . WILLIAMS,K. STROM-BEROand R. KROES, Cytoehemical and ultrastructural alterations associated with confluent growth in cell cultures of epithelial-like cells from rat liver. Lab. Invest. 29, 293 (1973). G . M . WILLIAMS,E. K. WEISBURGERand J. H. WEISBURGER,Isolation and long-term culture of epithelial-like cells from rat liver. Exp. Cell Res. 69, 106 (1971). G. YAsUZUMIand R. SUOIHARA,The fine structure of nuclei as revealed by electron microscopy. The fine structure of Ehrlich ascites tumor cell nuclei in preprophase. Exp. Cell Res. 37, 207 (1965). C. BOg~K, Neoplastic transformation in vitro of a clone of adult liver epithelial cells into differentiated hepatoma-like cells under conditions of nutritional stress. Proc. nat. Acad. Sci. (Wash.), 69, 956 (1972). Y. OSHIRO, L. E. GERSCHENSONand J. A. DIPAoLO, Carcinomas from rat liver cells transformed spontaneously in culture. Cancer Res. 32, 877 (1972). J. SATO, M. NAMBA,K. UsuI and D. NAOANO, Carcinogenesis in tissue culture. VIII. Spontaneous malignant transformation of rat liver cells in long-term culture. Jap. d. exp. Med. 38, 105 (1968). H. KATSUTAand T. TAKAOKA,Carcinogenesis in tissue culture. XIV. Malignant transformation of rat liver parenchymal cells treated with 4-nitroquinoline 1-oxide in tissue culture, d. nat. Cancer Inst. 49, 1563 (1972). K. KOSHIBA,M. NAMBAand T. ODA, Electron microscopic studies on cultured rat liver cells transformed by 4-nitroquinoline 1-oxide. Gann 61, 233 (1970). J. SATO,Malignant transformation in culture of rat liver cells treated with or without 3'-methyl-4-dimethylaminoazobenzene. In Cancer Cells in Culture. (Edited by H. KATSUTA) p. 335. University of Tokyo Press, Tokyo (1968). G . M . WILLIAMS,J. M. ELLIOTTandJ. H. WEISBUROER,Carcinoma after malignant conversion in vitro of epithelial-like cells from rat liver following exposure to chemical carcinogens. Cancer Res. 33, 606 (1970). C. ALTANER and E. HLAVAYOVA,Transformation of rat liver cells with chicken sarcoma virus B77 and murine sarcoma virus, d. Virol. 11, 177 (1973). K . K . SANFORD, Biologic manifestation of oncogenesis in vitro: A critique. J. nat. Cancer Inst. 53, 1481 (1974). S. KARASAKI,The fine structure of proliferating cells in preneoplastic rat livers during azo-dye carcinogenesis. J. Cell Biol. 40, 322 (1969). S. KA~ASAKI, Subcellular localization of surface adenosine triphosphatase activity in preneoplastic liver parenchyma. Cancer Res. 32, 1703 (1972). S. KARASAKI,Cell proliferation and subcellular localization of alkaline phosphatase activity in rat liver during azo dye carcinogenesis. Cancer Res. 35, 482 (1975).
533
S. Karasaki et al.
534
25.
26. 27.
28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46.
L.P. MERKOW,S. M. EPSTEIN, E. FARBER,M. PARDOand B. BARTAS,Cellular analysis of liver carcinogenesis. III. Comparison of the ultrastructure of hyperplastic liver nodules and hepatocellular carcinomas induced in rat liver by 2-fluorenyl acetamide. J. nat. Cancer Inst. 43, 33 (1969). I.B. WEINSTEIN, R. GEBERT, V. C. STADLER,J. M. ORENSTEINand R. AXLE, Type C virus from cell cultures of chemically induced rat hepatomas. Science 178, 1098 (1972). A . B . NOVlKOFF and S. GOLDFISHER,Visualization of peroxisomes (microbodies) and mitochondria with diaminobenzidine. J. Histochem. Cytochem. 17, 675 (1969). M. WACHSTEIN and H. MEISEL, Histochemistry of hepatic phosphatase at physiologic pH. Amer. J. din. Pathol. 27, 13 (1957). Z. HRUBAN, E. L. VIGIL, A. SLEVERS and E. HOPKINS, Microbodies--constituent organelles of animal cells. Lab. Invest. 27, 184 (1972). A. MARTINEZ-PALOMO,C. BRAISLOVSKYand W. BERNHARD, Ultrastructural modifications of the cell surface and intercellular contacts. Cancer Res. 29, 925 (1969). W. BERNHARD, The detection and study of tumor viruses with the electron microscope. Cancer Res. 20, 712 (1960). D . G . CHASEand L. P. PII~O, Expression of A- and C-type particles in early mouse embryo. J. nat. Cancer Inst. 51, 1971 (1972). C. ROUILLER and A. M. JEZEQUIEL, Electron microscopy of the liver. In The Liver. Morphology, Biochemistry, Physiology. I. (Edited by C. ROUILLER) p. 195. Academic Press, New York (1963). E.B. THOMPSON, G. M. TOMKINSand J. F. CURRAN, Induction of tyrosine-aketo glutarate transaminase by steroid hormones in a newly established tissue culture line. Proc. nat. Acad. Sci. (Wash.) 56, 296 (1966). W. OEnLERT, Cellular proliferation in carcinogenesis. Cell Tissue Kinet. 6, 325 (1973). A. SIMARD,G. COUSINEAUand R. DAOUST,Variations in the cell cycle during azo dye carcinogenesis. J. nat. Cancer Inst. 41, 1257 (1968). S. KARASAKI, Virus particles associated with the transplantable Novikoff hepatoma in the rat. Cancer Res. 29, 1313 (1969). J. LOCKER, P. J. GOLDBLATTand J. LEIGHTON,Some ultrastructural features of Yoshida ascites hepatoma. Cancer Res. 28, 2039 (1968). A . B . NOVIKOFF and L. BIEMPICE, Cytoehemical and electron microscopic examination of Morris 5123 and Reuber H-35 hepatomas after several years of transplantation. Gann Monogr. 1, 65 (1966). S. KARASAKI, An electron microscope study of intranuclear canaliculi in Novikoff hepatoma cells. Cancer Res. 30, 1736 (1970). E. BUCCIARELLI, Intranuclear cisternae resembling structures of the Golgi complex. J. Cell Biol. 30, 664 (1966). H. HANAOKA and B. FRIEDMAN, Paired cisternae in human tumor cells. J. Ultrastruct. Res. 32, 323 (1970). R . O . KELLY,Ultrastructural comparisons of paired cisternae in leukemic and mesenchymal cells. Anat. Rec. 171, 559 (1971). R . a . KESSEL,Annulate lamellae. J. Ultrastruct. Res. Suppl. 10, 1 (1968). E. ESSNER and M. D. REUBER, Localization of acid phosphatase activity in well-differentiated hepatocellular carcinoma 146. J. nat. Cancer Inst. 479 25 (1971). S. KARASAKI, Surface membrane ATPase activity in transformed rat liver epithelial cell lines. J. Cell Biol. 679 200a (1975).