Cytological observations of “nonproducer” Rous sarcoma cells

VI~O~~~GT 27, 360-37’7 (1965)

Cytological

Observations HENRY

Departments

of Anatonzy

of “Nonproducer”

S. DI STEFANO

AND

ROBERT

Rous Sarcoma M. DOUGHERTY”

and Microbiology, State University of Xew York, Upstate Medical Syracuse, flew York Accepted July

Cells’

Center,

29, 1965

“Nonproducer” cells, obtained by infection of cultivated chick embryo fibroblasts with ROW sarcoma virus (Bryan strain), were compared and contrasted with similarly infected cells that were producing virus in large quantities and with uninfected control cells. Both types of infect.ed cells differed markedIy from uninfected cells but, despite quantitative differences between them, the morphology of the two types of infected cells was essentially the same. Inasmuch as the “nonproducer” cells did not involve infection with the “helper” Rous-associated virus, the alterations observed reflect,ed only infection with Rous sarcoma virus. INTRODUCTION

Hanafusa et al. (1963) have demonstrated that certain strains of Rous sarcoma virus (RSV) are defective, in that virus product’ion depends upon dual infection with RSV and a second “helper” virus which has been termed Rous-associat,ed virus (RAV) by Rubin and Vogt (1962). As a consequence, most investigations of RSV have been done with virus stocks that contained two distinct agents. It is apparent, therefore, t*hat studies of defective RSV should take into account two factors, RSV and RAV. Previous descriptions of cellular alterations resulting from infection with RSV did not distinguish between effects induced by RSV per se and those of the associated virus. Infection of chick embryo fibroblasts in vitro with either RAV or visceral lymphomatosis virus (VLV) did not produce significant morphologic alterations in the cells aside from virus production at the cell surface. (Di Stefano and Dougherty, unpublished 1Supported by grants PN-5593 from the National Science Foundation and E-328 from the American Cancer Society. 2 The authors wish to acknowledge the technical assistance of Mrs. Ursula Feller and Miss Joan Long.

observations), However, t.his did not exclude the possibility that either of these agents had an influence on cell morphology when present in combination with RSV. Under conditions of solitary infection of chick cells with RSV, where superinfection with helper is excluded, “nonproducer” (NP) cells appear, which are morphologically altered but fail t’o produce infectious virus. However, Dougherty and Di Stefano (1965) have demonstrated the production of noninfectious virus particles by these cells. This investigation was undertaken to ascertain the morphological characteristics of NP cells which would, of necessity, reflect alterations due to infection wit.h RSV alone. MATERIALS

AND

METHODS

The experimental procedures used in this study are described in the preceding report (Dougherty and Di Stefano, 1965). RESULTS

AND

DISCUSSION

Cytological aIterations resuIting from dual infection of cultivated chick embryo cells with RSV and RAV have been reported, both at the level of the light microscope (Halberstaedter et al., 1941; Doljanski and Tenenbaum, 1942; Tenenbaum and Dol3GO

L‘NONPRODUCER”

ROUS SARCOMA

janski, 1943; Lo et al., 1955; Temin and Rubin, 1958; Gold&, 1959; Rubin, 1960; Hampton and Eidinoff, 1962) and with the electron microscope (Haguenau, 1960; Haguenau and Beard, 1962). The observations presented in this report demonstrate that Rous sarcoma cells in tissue culture exhibit striking structural alterations when compared to uninfected chick embryo fibroblasts and that comparison of “producer” (P) and “nonproducer” (NP) transformed cells, while revealing some quantitative differences shows the two cell types to be basically similar in appearance. General Topography One of the earliest and most conspicuous alterations observed after infection with RSV in tissue culture has been the transformation of fusiform fibroblas t-like cells into rounded ones (Halberstaedter et al., 1941; Doljanski and Tenenbaum, 1942; Tenenbaum and Doljanski, 1943). Our electron microscope examination of P cells were in agreement with the earlier light microscope observations, and the same effect was observed as an early change in the transformed infected NP cells. At low levels of magnification, uninfected control cells were long, slender, and fusiform in shape (Figs. l-3), while sections of NP cultures (Figs. 4 and 5) and P cultures (Figs. 6-8) revealed a transformation to the rounded cell type. Further, sections of NP cells demonstrated the presence of many small cytoplasmic processes extending from the main body of the cells, interdigitations between the plasma membranes of adjacent cells and many regions of close intercell contact (Figs. 4 and 5), which were also seen in P cells (Figs. S-S), but not in uninfected cells. Changes in nuclear profiles were a second general topographical modification observed in cultures of Rous-infected cells. In the case of NP cells, the nuclear envelopes appeared consistently to be thrown into many deep folds resulting in lobulated and otherwise bizarrely shaped nuclei (Figs. 4, 5, and 9-l 1). Infolding of the nuclear envelope was also seen, but witlh less frequency, in sections of P cells. Here, profiles of some nuclei appeared unaltered (Figs. 12 and 13), whereas

CELLS

361

others (Figs. 7 and 14-16) resembled nuclei of NP cells. This feature has been observed frequently in nuclei of malignant cells in general (Oberling and Bernhard, 1961) and in nuclei of Rous cells (Haguenau, 1960) ; it is believed to result from nuclear hypertrophy. With respect to cell and nuclear profiles, it should be pointed out that changes in these parameters resulted in a rather uniform population of NP cells while a great deal more variation, particularly in nuclear shapes, was observed in sections of P cells. Doljanski and Tenenbaum (1942) described variations in shapes of virus-producing cultivated Rous sarcoma cells, representing transitional forms between the fibroblast-like cells and the rounded cells, and suggested that the transformation was a reversible one. The apparent difference between the uniformity of shapes seen in cultures of NP cells and the variations noted in cultures of P cells could be accounted for if one assumed that the transformation of NP cells was an irreversible process and that the portions of NP cultures examined contained no transitional forms. Another explanation for this difference between the two types of Rousinfected cells might be a consequence of the fact that the NP cultures were the progeny of a single infected cell clone, while P cultures were induced by several cycles of infection and therefore represented the progeny of many virus-cell interactions. Topographical comparisons also revealed a greater degree of electron density in the cytoplasm of NP cells (Figs. 4, 5, and 9-11) when compared to that of uninfected cells (Figs. l-3) where the cytoplasmic material appeared consistently “paler.” In this respect the NP cells bore a closer resemblance to the P cells (Figs. 6-8 and 12-16). The presence of a highly developed “vacuolar system,” dense osmiophilic inclusions, and myelin figures in the cytoplasm of Rous cells has been described by Haguenau and Beard (1962). In this investigation, uninfected cells were found to contain some electron-lucent vesicles in the cytoplasm (Figs. 1 and 2). Our observations of P cell cultures agreed with those of Haguenau and Beard (1962) in that they revealed the presence of many electron-lucent vesicles of various dimensions and many

362

DI STEFANO

FIGS. l-3. Uninfected chick embryo X 6000; Fig. 3, X 10,500.

fibroblast,s

AN11 DOUGHERTY

grown in vitro. Magnification:

Fig. 1, X 7100: FIG. 2

“NONPRODUCER”

FIG. 4. RSV-infected

ROUS SARCOMA

KP cells. Magnification:

CELLS

X 15,000.

363

364

DI STEFANO

FIG. 5. RSV-infected

AND

DOUGHERTY

NP cells. Magnification:

X 16,700.

“NONPRODUCER”

FIGS. 6-8. RSV-infected

P cells. Magnification:

ROUS

SARCOMA

CELLS

365

Fig. 6, X 8200; Fig. 7, X 36()13; Fig. 8, x fL300.

366

DI STEFANO

AND DOUGHERTY

FIGS. 9-11. Bizarre-shaped nuclei of RSV-infected NP cells. Magnification: Fig. 9, X 9100; Fig. 10, X 12,200; Fig. 11, X 6200. FIGS. 12-16. Nuclei of RSV-infected P cells showing variation in shapes. Magnification: Figs. 12 and 13, X 7700; Figs. 14 and 15, X 6500; Fig. 16, X 10,900.

“NONPRODUCER”

ROUS

SARCOMA

CELLS

367

FIG. 17. RSV-infected P cells showing evidence of cell degeneration. Magnification: X 6700.

osmiophilic inclusions (Figs. 6-8 and 17). These cultures however, also demonstrated general cell da,mage as evidenced by the presence of degenerating cells and cell debris among otherwise intact cells (Fig. 17). Cytopathic changes of this nature were also noted by Lo et al. (1955) in Rous-infected chicken fibroblasts. The development of a vacuolar system was also found to be a feature of NP cells (Figs. 4, 5, and 9-11). In NP cells, however, the variation in size and number of vesicles was not as pronounced as in P cells, and in contrast to the cell destruction observed in cultures of P cells, little or none was evident in cultures of NP cells. This correlated with the fact that heavily infected P cells could seldom be carried for more than 3 or 4 transfers, whereas NP cells were maintained without difficult’y for at least’ 8 passages. Nucleus Further similarities between the two types of Ron-infected cells studied were evidenced by comparison of P cell intranuclear morphology with that of NP cell nuclei. While the nuclear material of uninfected cells was finely granular and had a

rather homogeneous distribution (Figs. l-3), that of NP cells frequently revealed the presence of coarse chromatin clumps (Figs. 4, 5, and 9-11) similar in appearance to that seen in P cells (Figs. 6-8 and 12-16) and occasional evidence of chromatin margination (Figs. 9, 18, and 19). Some increase in nucleolar size was observed in nuclei of NP cells, but this phenomenon was more characteristic of nuclei in P cells (Figs. 8, 12, and 13). A frequent observation that was common to both NP (Figs. 18-20) and P (Figs. 2124) cell nuclei, but never seen in nuclei of uninfected cells, was the presence of regular and/or irregular dense bodies that were sometimes associated with nucleolar material. These were resolved as aggregates of particles (white snows) measuring 250-300 A3 and were also seen as disassociated masses of particles (black arrows). In appearance and location these nuclear inclusions apparently correspond to the dense bodies previously described by Haguenau (1960) in tumor cell nuclei of viral origin. The nuclear features presented herein 3Ribosomes in these cells measured approximately 150 A.

368

FIGS. 18-20. Dense osmiophilic

DI STEFANO

AND

DOUGHERTY

bodies in nuclei of RSV-infected

XP cells. Magnificatioll

: X 75,GOU.

FIGS. 21-24. Dense osmiophilic bodies in nuclei of RS-infect.ed FIG. 25. Cytoplasmic region in uninfected control cell showing plasm, and paucity of free ribosomes. MagnificaGon: X 34,700.

P cells. Magnification: X 75,600. mitochondrion, organized ergasto-

370

DI STEFANO

AND

have been pointed out by Haguenau (1960) as possibly being associated with the infective agent. Except for the fact that nucleolar hypertrophy was less pronounced in nuclei of NP cells than in those of P cells, the intranuclear alterations seen in the P cell nuclei were duplicated in nuclei of NP cells. Thus, if indeed these changes in P cells were indicative of a relationship to the infecting agent, then the NP cells showed the same relationship.

DOUGHERTY

number of mitochondria (Figs. 4 and 5) which were often found in aggregates (Figs. 29 and 30). Further, the mitochondria of NP cells were frequently atypical in shape (Figs. 29-31), very large (Fig. 32) and sometimes exhibited alteration of internal morphology, evidenced by disruption of cristae and the presence of swollen regions (Figs. 4, 5, and 29-31). An increased mitochondrial population was also seen in P cells (Figs. 6 and 7), but mitochondrial aggregates and atypically shaped forms were not observed. Cytoplasm Disruption of internal morphology was more The cytoplasm of NP, P, and uninfected extensive and was seen more frequently in P cells contained the organelles generally cells (Figs. 33 and 34) than in NP cells. found in cells. Differences and similarities There has been some controversy as to were observed among the 3 types of cells whether or not the mitochondrial population of malignant cells increases, decreases, or studied and these were, for the most part, quantitative in nature. remains unchanged. On the basis of mitoRibosomal constituents. Sections of uninchondrial counts in rat liver tumors reported fected control cells showed the presence of by Allard et al. (1952) and by Shelton et al. (1961) an organized ergastoplasm (Figs. 3 and 25), (1953), Oberling and Bernhard stated that malignant cells generally conas well as the presence of free ribosomes that were seen either singly or in small clusters tained reduced numbers of mitochondria of smaller size when compared to nonmalignant (Fig. 25). In NP cells, this cytoplasmic cells. Reports also indicated that, generally, complex showed a marked quantitative alteration of mitochondrial morphology is a difference (Figs. 26 and 27). Although little tumor cells; t.he change was noted in the organized ergasto- feature of virus-induced plasm, there was an increase in the free most common change is “cloudy swelling” ribosomal constituents, which were seen as (Bernhard and Oberling, 1957; Oberling, 1959; Roullier, 1960; Haguenau, 1960). single particles, in clusters and in interWith respect to Rous virus per se, reports of connected polysomal-like chains randomly alterations in mit’ochondrial number after distributed within the confines of the cytoRous virus infection are also contradictory. plasm. It was probably this increase that Gaylord (1955), Bernhard et al. (1956), and imparted the increased cytoplasmic density seen at lower magnifications (Figs. 4 and 5). Epstein (1957) reported decreases in the population of infected cells In this, respect, the appearance of NP cells mitochondrial was very similar to that of P cells (Figs. whereas Gold6 (1959) noted a more highly developed mitochondrial system and Hamp6-8 and 28). These observations agree with ton and Eidinoff (1962) observed no apthose of Haguenau (1960) for virus-producing Rous-infected cells and are the basis for preciable differences. The results presented in this paper inreports of increased basophilia (Doljanski and Tenenbaum, 1942; Tenenbaum and dicated an increase in mitochondrial numDoljanski, 1943; Temin and Rubin, 1958; bers in both types of Rous-infected cells as well as the presence of mitochondrial swellGolde,, 1959; Vigier and Gold& 1959; Rubin, 1960; Hampton and Eidinoff, 1962) and in- ing. The latter feature, however, was more prevalent in mitochondria of P cells, while creased ribonucleic acid synthesis (Rubin, 1960; Hampton and Eidinoff, 1962) in ROUS the presence of enlarged and abnormally shaped mitochondria was a more frequent sarcoma cells. observation in the cytoplasm of NP cells. Mil.ochondria. The mitochondria of uninGolgi complex. The Golgi complex was fected cells were relatively few in number another organelle that showed a consistent (Figs. l-3) and had the classical morphology change in the (Fig. 25). NP cells showed an increase in and striking quantitative

“NONPRODUCER”

ROUS

SARCOMA

CELLS

371

FIGS. 26 and 27. Cytoplasmic regions in RSV-infected NP cells showing large numbers of free ribosomes. Magnification: X 67,500. FIG. 28. Cytoplasmic region in RSV-infected P cell showing large numbers of free ribosomes. Magnification: X 75,600.

cytoplasm of NP cells. A modest but classical Golgi was seen in most uninfected cells examined (Fig. 35, arrows), and some indication

of

Golgi

hyperplasia

was

observed

in

the cytoplasm of P cells (Fig. 36, arrows). In contrast to this, nearly all NP cells examined showed large quantities of Golgi material in the cytoplasm, and the increase

FIGS. 29 and 30. Mitochondrial aggregates in cytoplasm of ICSV-infected NP cells. Magnification: pig. 29, X 23,500; Fig. 30, X 31,300. FIG. 31. Atypically shaped mitochondrion in cytoplasm of RSV-infected NP cell. Magnification: x 54,400. FIG. 32. Giant mitochondrion (approximately 12 p) in cytoplasmic process of a RSV-infected NP cell. Magnification: X 20,400. 372

“NONPRODUCER”

ROUS

SARCOMA

CELLS

FIGS. 33 and 34. Morphological alteration of mitochondria in RSV-infected P cells. Magnification: Fig. 33, X 14,400; Fig. 34, X 46,900. FIG. 35. Golgi zone in cytoplasm of uninfected control cell. Magnification: X 52,900. FIG. 36. Golgi zone in cytoplasm of RSV-infected P cell. Magnification: X 48,460.

373

374

111 STEFA?r’O

F 'IGS. 37 and 38. Golgi zones in cytoplasm Fig. 38, X 39,700.

AND

IIOIJGHERTY

of RSV-infected

NP cells. Mitgnification:

Fig. 37, X 45

FIG. 39. Particles resembling incomplete virus particles (arwws) in ryt,opl:tsm of I:SV-illfected P cell. Magnification: X 115,700. FIG. 40. A particle (arrow) similar to those seen in Fig. 39, in cytoplasm of HST’-infected NP cell. Magnification: X 115,700. FIG. 41. Aggregation of vesicular structures (CUTOWS)in cytoplasm of l:SIT-iltfected NP cell. JI:tgnification: X 115,700. 375

376

Dr STEFANO

AND DOUGHERTY

was particularly evident in the vesicular component (Figs. 29, 37, 38, arrows). Our observations, therefore, indicated a quantitative difference in Golgi between the two types of Rous-infected cells examined; Golgi hyperplasia was found to be a prominent feature of NP cells but not of P cells. Other reports of quantitative changes in Golgi are contradictory. Tenenbaum and Doljanski (194311, Haguenau (1960), and Haguenau and Beard (1962) reported the presence of Golgi hyperplasia in virus-producing Rous sarcoma cells, whereas Bernhard et al. (1956) stated that the Golgi was most often underdeveloped or absent.

Cytop%asmic Particles Clusters of intracytoplasmic particles resembling incomplete virus particles were seen frequently in RSV-infected P cells (Fig. 39). They contained an electron-lucent

central1 core measuring approximately 43 rnp and at single external electron-dense component

which

gave

the

entire

particle

a

dimen.sion of 60 rnp. These were similar in structure, location, and dimensions to particles described by Haguenau et al. (1962) in cells of chick embryo chorioallantoic membrane infected with RSV. If these particles bore any relationship to Rous infection, as suggested by Haguenau et al. (1962), then their presence in NP cells could be interpreted in a similar manner. Examination of NP 1~11sdid indeed reveal the presence of several such particles. One particle whose morphology was identical to that of those observed in the P cells is shown in Fig. 40,

another feature between these The production cells lhas been

demonstrating the similarity two types of infected cells.

of noninfectious virus by NP reported by Dougherty and Di St,efano (1965). The quantities of virus produ.ced by NP cells was low compared to

that produced by P cells. Thus, the numbers of intracellular particles seen in P and NP cells might be a reflection of these differences in virus production. In one other instance, an aggregation of particles with a diameter of approximately 52 m/L was seen in the cytoplasm of an NP cell (Fig. 41). These did not resemble the “incomplete viruslike particles” that were

seen in the cytoplasm of P cells. The significance of these particles, or whether they are related to viral infection, is not known, nor is any hypothesis offered. However, it is

interesting to note that their dimensions agree with those of that portion of mature

ROUSvirus which is bounded

by the inter-

mediate membrane. CONCLUSION Despite some differences, the morphology of P and NP Rous sarcoma cells wa,sessentiaIly similar. The NP cells were the result of solitary infection with RSV, while the P cells arose from dual infection with RSV and “helper virus.” The alterations in NP cells described in this paper, therefore, can be attributed t.o Rous virus per se, free of the influence of the helper. Thus, it is possible to state that the morphology of the transformed Rous sarcoma cell at the fine structural level, which has been described in this report, is a helper-independent property of Rous virus. REFERENCES ALLARD, C., MATHIEU, R., DE LAMIRXNDE, G., and CANTERO, A. (1952). Mitochondrial popu-

lation in mammalian cells. I. Description of a counting technique and preliminary results on rat liver in different physiological and pathological conditions. Cancer Res. 12, 407412. BERNHARD, W. (1958). Electron microscopy of tumor cells and tumor viruses. Cancer Res. 18, 491-509. BERNHARD, W., and OBERLING, C. (1957). Electron microscopy of the malignant cell with special references to viruses. Can. Cancer Conf. 2, 59-81. BERNHARD, W., OBERLING, C., and VIGIER, P. (1956). L’ultrastructure de particules virus du sarcome de Rous et leur rapport avec le cytoplasme des cellules tumorales. BUZZ. Cancer 43, 407-422. DOLJANSKI,

L., and TENENBAUM, E. (1942). Studies on Rous sarcoma cells cultivated in vitro. I. Cellular composition of pure cultures of Rous sarcoma cells. Cancer Res. 2, 776-785. DOUGHERTY, R.M.,and DI STEFANO, H.S. (1965). Virus particles associated with “nonproducer” Rous sarcoma cells. Virology 27, 351-359. EPSTEIN, M. A. (1957). The fine structural organization of the Rous tumor cells. J. Biophys. B&hem. Cytol. 3, 851-858. GAYLORD, W. H. (1955). Virus-like particles associated with the Rous sarcoma as seen in sections of the tumor. Cancer Res. 15, 80-83.

"~;OKPKODUCER"

ItOW

A. (1959). etude quantitative des modifications cellulaires provoquees par le virus du sarcome de Row dans les cultures des cellules embryonnaires de poulet. Exptl. Cell Res. 18, 528-541. HAGUENAU, F. (1960). Significance of ultrastructure in virus-induced tumors. Natl. Cancer Inst. Monograph 4, 211-247. HAGUENAU, F., and BEARD, J. W. (1962). The avian sarcoma-leukosis complex; its biology and ultrastructure. In “Ultrastructure in Biological Systems” (A. J. Dalton and F. Haguenau, eds.), Vol. 1: “Tumors Induced by Viruses; Ultrastructure Studies,” pp. 1-59. Academic Press, New York. HAGUENAU, F., FEBVRE, H., and ARNOULT, J. (1962). Mode de formation intracellulaire du virus du sarcome de Rous. Etude ultrastructurale. J. Microscopic 1,445454. HALBERSTAEDTER, L., DOLJANSEI, L.,and TENENBAUM, E. (1941). Experiment,s on the cancerization of cells in vitro by means of Rous sarcoma agent. Brit. J. Exptl. Pathol. 22, 179-187. HAMPTON, E. G., and EIDINOFF, M. L. (1962). Transformation of chick embryo cells in culture by Rous sarcoma virus-cytochemical studies. Cancer Res. 22, 106-1066. HbNAFUSA, H., HANAFUSA, T., and RUBIN, H. (1963). The defectiveness of Rous sarcoma virus. Proc. Natl. Acad. Sci. U.S. 49, 572680. Lo, W. H. Y., GEY, G. O., and SHAPRAS, P. (1955). The cytopathogenic effect of the Rous sarcoma GOLDS,

SARCOMA

CELLS

377

virus ou chicken fibroblasts in tissue culture. Bull. Johns Hopkins Hosp. 96, 248-266. OBERLING, C. (1959). The structure of cytoplasm. Intern. Rev. Cytol. 8. l-32. OBERLING, C., and BEENHARD, W. (1961). The morphology of cancer cells. In “The Cell” (J. Brachet and A. E. Mirsky, eds.), Vol. 5, pp. 405-496. Academic Press, New York. ROULLIER, C. (1960). Physiological and pathological changes in mitochondrial morphology. Intern. Rev. Cytol. 9, 227-292. RUBIN, H. (1960). An analysis of the assay of Rous sarcoma cells in vitro by the infective center technique. Virology 10, 2949. RUBIN, H., and VOGT, P. K. (1962). An avian leukosis virus associated with stocks of Rous sarcoma virus. Virology 17, 184-194. SHELTON, E., SCHNEIDER, W. C., and STRIEBICH, M. J. (1953). A method for counting mitochondria in tissue homogenates. Exptl. Cell Res. 4, 3241. TEMIN, H. M. and RUBIN, H. (1958). Characteristics of an assay for Rous sarcoma virus and Rous sarcoma cells in tissue culture. Virology 6, 669-688. TENENBAUM, E., and DOLJANSKI, L. (1943). Studies on Rous sarcoma cells cultivated in vitro. II. Morphologic properties of Rous sarcoma cells. Cuncer Res. 3, 585-603. VIGIER, P., and GOLD& A. (1959). Growth curve of Rous sarcoma virus on chick embryo cells in vitro. Virology 8, 60-79.