- Email: [email protected]
Factors influencing the determination of cellular morphology in cells infected with Rous sarcoma virus
VIROLOGY
18,
Factors
524-534 (1962)
Influencing in Cells
the
Determination
Infected
with
ALFRED Lkpartment
of Pathology, New
Rous
Sarcoma
Morphology Virus’
M. PRINCE2
Yale Haven,
Accepted
of Cellular
University Connecticut July
School
of Medicine,
23, 1962
Additional means have been described for controlling the presence or absence, and nature, of morphological transformation induced by Rous sarcoma virus infection of turkey fibroblasts in vitro. Eagle’s medium supplemented with serum from various species was found to support only limited morphological transformation under normal circumstances. The addition of 10% tryptose broth to such media markedly favored the production of morphological transformation. Infection of cells at various times after a 24-hour period of starvation in serum-free Eagle’s solution without glucose and glutamine at room temperature resulted in marked variations in both the total extent of morphological transformation and the nature of the transformation which was observed. Tumors induced in viva by single virus particles varied widely in morphology. Tumors induced in this manner having predominantly spindle-cell, giant-cell, fusiformcell, and round-cell morphologies were observed. The above findings are discussed particularly in reference to possible correlations between in vitro and in viva morphology, and in vitro morphological conversion with neoplastic potential; and possible mechanisms underlying the production of morphologic conversion by Rous sarcoma virus. INTRODUCTIOK
It is a unique property of the Rous sarcoma virus that cells maintained in vitro may be rapidly converted by infection into cells having various types of altered morphology. The most common alteration is the appearance of the round basophilic cells first described by Halberst,aedter et al. (1941). The conversion of cells to this type of morphology was shown by Manaker and Groupi! (1956) to provide a means for in vitro assay of Rous sarcoma virus, since under suitable condtions these cells proliferated into discrete countable foci. l These studies were supported by research grants from the American Cancer Society, and from the Jane Coffin Childs Fund for Medical Research. ‘Present address: Wistar Institute, Philadelphia, Pennsylvania.
In 1960 Temin report,ed additional types of morphological conversion and analyzed some of the factors determining the production of alternate morphological responses. It was found that virus derived from certain clones had the property of converting cells to long spindle-shaped morphology; and that this ability reflected a genetic property of the virus. On the other hand, isolation of virus clones from foci having giant-cell or fusiform-cell morphology revealed that this response did not depend on the genetic nature of the infecting virus, since these clones produced primarily round-cell foci. It appeared that the production of giantcell and fusiform alteration depended on the physiological state of the cells at the time of infection. Temin (1960) also has reported that RSV-infected cells may undergo no mor-
524
DETERMINATION
OF CELLULAR
phological alteration. This was observed when infected monolayers were not fed at regular intervals and was also observed in the infection of a cloned line of fibroblasts which had been maintained for 3 months in vitro prior to infection. With these cells the number of foci of morphologically transformed cells was far below the number of cells shown to have been infected by infectious center assays. That not all virus-infected cells show morphological transformation may also be concluded from the finding that in certain tissue culture media only a small fraction of RSV-producing cell clones can be morphologically distinguished from uninfected control clones (Prince, 1960b) ; and from the finding that fetal calf serum can suppressthe appearance of foci of morphologically altered cells following infection of monolayers with small amounts of RSV, but does not change the proportion of infected cells (Rubin, 1960). The above studies raise the important and so far inadequately answered question as to what correlation exists between in vitro morphology and in viva behavior. It, is of interest, in this regard that inoculation of virus stocks having genetic markers for round-cell or spindle-cell conversion resulted in t’umors which were morphologically indistinguishable (personal communication, I-I. M. Temin). The present, report will present evidence that the morphological responsecan be controlled in vitro by choice of tissue culture media, and by infecting cells at various times after preconditioning. In addition, it will be shown that tumors produced in viva by single virus particles may vary significantly in morphology and that the morphological variations observed in viva appear to correspond t,o those which have been dcscribed to occur in vitro.
MORPHOLOGY
525
assay was by the chorioallantoic membrane technique (Prince, 1958). Histological Procedures Tissue cult,ures for morphological examination were grown on coverslips in Leighton tubes. Coverslips were fixed in absolute met.hanol and stained by the MayGriinwald Giemsa procedure. Tumor tissues were fixed in Bouin’s solution and stained with hematoxylin and eosin. Procedure for Infection of Cultures at Various Times after Preconditioning
Turkey fibroblests cultivated in Eagle’s medium containing 5% calf and 5% turkey serum were permitted to grow unt,il approximately one-half of the surface of the glass had been covered. At this time cultures were washed with “deficient medium” (Eagle’s solution lacking serum, glucose, and glutamine). A proportion of the cultures were then fed with t.he “deficient medium” and held for 24 hours at room temperature. A second group of cultures was fed with complete growth medium and held for 24 hours at 37°C. At the end of t,he 24-hour preconditioning period, a group of cultures was immediately infected by addition of approximately 1 plaque-forming unit (PFU) per cell of Bryan strain Rous sarcoma virus suspended in 0.2 ml of complete medium. The remaining cultures were fed with complete medium. After 5 additional hours at 37”, infected cult,ures were washed four t,imes with PBS and refed with complete medium. At t,his time a second group of cultures was infected as above and allowed to remain in the presenceof the virus inoculum for a 5-hour period. Addit,ional cultures were infected for the periods 10-15, 15-20, and 20-25 hours following preconditioning. Similar groups of cultures were exposed to inocuia lacking virus but were otherwise MSTERIALS AND METHODS treated as described above. All cultures were Virus strains, and tissue culture proce- fed with complete growth medium 48 hours dures, employed in this study have been after termination of the preconditioning previously described (Prince, 1960a). The period. Cultures were stained for histological exchickens employed were from the Cornucopia (“Pure Hope”) line of White Leghorns amination, and supernates removed for virus asSay, 72 or 96 hours after infection. previously described (Prince, 1958). Virus
PRINCE
526
ever, characteristic basophilic round cells were extremely rare. Similar results were Effect of Maintenance Medium on Morphoobtained with Eagle’s media containing all logical Responseof Turkey Fibroblasts the serum types and combinations described to Infection by RSV in Vitro above when tryptose phosphate broth was It was a puzzling fact that in the course not present. In contrast to these results, of three years of study of various aspects cultures overlaid wit,h tryptose phosphate broth containing medium showed conversion of the infection of chick and turkey fibroof the majority of the cells in the culture blasts by RSV in vitro, morphological transto intensely basophilic round cells (Fig. formation of the type observed by Manaker and Group&, and Rubin and Temin, had 1B). Assays carried out on supernatant fluids never been observed. This was the case even with Bryan strain virus, with which it could from sister cultures to those portrayed in be shown that the majority of the cells in Fig. 1, taken 70 hours after infection, rethe culture rapidly became converted into vealed 106.6 PFU/ml in cultures overlaid virus producers (Prince, 1960a). The media with ET&, and 105.0PFU/ml in cultures employed in these st,udies (in which mor- overlaid wit,h the tryptose phosphate broth phological conversion did not occur) were containing medium. There were thus cerEagle’s basal medium containing either tainly no fewer virus producing cells in 10% duck serum (ED,,), 10% turkey serum cultures overlaid with media lacking tryp(ET,,), 10% calf serum (ECi,) , 5% duck tose phosphate, in which morphological serum plus 5% calf serum (ED&), or 5% transformation did not occur. turkey serum plus 5% calf serum (ET&s). It appeared likely that the absence of mor- Effect of Preconditioning on Morphological Response of Fibroblasts to Infection by phological conversion was a consequence of Rous Sarcoma Virus the difference in the media employed in these studies as compared to the media emSeveral experiments were carried out to ployed in studies in which morphological determine the effect of infecting fibroblasts conversion was observed. The most obvious at various time int.ervals after preconditiondifference between such media was the ing in an attempt to produce a degree of association of tryptose phosphate broth con- biochemical synchrony. Table 1 summarizes taining media with the presence of morpho- the results of one such experiment in which logical conversion. tenth passage turkey fibroblasts were inExperiments were therefore carried out fected at an input multiplicity of 1 PFU per in which turkey fibroblasts were infected cell with Bryan strain virus at various times with Bryan strain virus at an input mult,iafter being held for 24 hours at room templicity of 10 PFU per cell. Cultures were perature in a deficient medium composed washed twice and overlaid with different of Eagle’s solution lacking serum, glucose, media after being held for 2 hours at 37” to and glutamine. It will be noted that this permit, virus adsorption. The result,s of an procedure did not significantly affect the experiment in which cultures were overlaid yield of virus in the supernate 4 days after with Eagle’s medium containing 5% calf infection. Other experiments, in which inand 5% turkey serum or with EagIe’s me- fectious center assays were done, similarly dium containing 10% duck serum and 10% failed to reveal significant variations. Howtryptose phosphate broth are presented in ever, microscopic examination of these culFig. 1. Infected cultures overlaid with me- tures 4 days after infection revealed that dium lacking tryptose phosphate broth the preconditioning procedure did have a were indistinguishable from cont,rols when marked effect on the degree of morphologiexamined without staining, 51 hours after cal transformation. Whereas control culinfection. In stained preparations there was tures, which had not been maintained in the noticeable increase in cytoplasmic baso- deficient medium, showed no significant philia in infected cultures (Fig. ID) ; how- degree of morphological transformation. RESULTS
DETERMINATION
OF CELLULAR
MORPHOLOGY
FIG. 1. Effect of maintenance medium on morphologic conversion of turkey fibroblasts b: Rous sarcoma virus (Bryan strain). (A) and (B) show turkey fibroblasts maintained in Eagle’s medium with 10% duck serum and 10% tryptose phosphate broth. Control uninfected cells are shown in (a); cells infected 51 hours previously in (B). (C) and (D) show turke\ fibroblasts maintained in Eagle’s medium wit.h 5% turkey and 5% calf serum. (C) shows control uninfected cells, and (D), cells infected with virus 51 hours previously. Magnification: x 315.
527
PRINCE
528
TABLE EFFECT
OF PRECONDITIONING INFECTION Preconditioning
prior
ON MORPHOLOGICAL BY RSV (BRYAN
‘emp. (“C)
RESPONSE “HIGH TITER”
-
to infection”
1st Incubation Medium
1
4 Days
2nd Incubation
-
rime (hr)
Medium -
Complete” Complete Complete Complete Complete
37 37 37 37 37
24 24 24 24 24
Complete Complete Complete Complete
Deficient* Deficient Deficient Deficient Deficient
21 21 21 21 21
24 24 24 24 24
Complete Complete Complete Complete
T ‘emp. w 37 37 37 37
-
Time (h-1 -
-I
after
PFU in supernate per cell in culture
FIBROBLASTS
infection
I
EEstimated yO ,:ells “transformed”d
I-
TO
Estimated “coverage” lIdday after transfer8
-
5 10 15 20
7.1 4.6 2.8 4.1 3.6
5 10 15 20
3.4 2.8 7.0 4.1 5.4
10
37 37 37 37
OF TURKEY STRAIN)
a Complete medium = Eagle’s with 5y0 calf and 57, duck serum. * Deficient medium = Eagle’s solution without serum, glucose, or glutamine. c Cultures were infected with 1 PFU/cell (input), washed for four times after a 5-hour adsorption, period, and refed complete medium. Cells were tenth passage turkey fibroblasts. “epithelioid” (fusiform) or giant morphology. d Transformed cells = cells having e Cultures trypsinized and subcultured 1:4 on fourth day after infection. “Coverage” refers to proportion of glass-surface covered by cells.
there was significant transformation to fusiform “epithelioid,” or giant cells, in cultures infected immediately after maintenance in deficient medium. Cultures infected between 5 and IO hours after refeeding following maintenance in deficient media resembled control cultures in that morphological transformation was minimal or absent. An especially marked degree of morphological transformation was noted in cultures infected between 15 and 25 hours after refeeding following maintenance in deficient medium. In this experiment cultures in each group were trypsinized 4 days after infection and divided 1 to 4 for subcultivation. When subcultures were examined 1 day after transfer, marked differences were readily noted in the degree of coverage of the glass surface by the subcultured cells. These differences corresponded to the degree of transformation previously observed. A large number of round cells were seen floating in the medium of the subcultures from the transformed
cultures. It is likely, since rounded cells are known to attach poorly to glass surfaces, that these cells were viable but differed from the untransformed cells in their ability to attach to the glass surface. The morphology of cells from an experiment which differed from the above only in that cultures were infected at 4-hour intervals following refeeding after maintenance in deficient medium, and that third passage turkey fibroblasts were employed, are presented in Fig. 2. Cells were examined 72 hours after infection. In this experiment the most marked degree of transformat,ion to basophilic round cells occurred in the cultures infected immediately after maintenance in deficient medium. It was observed, however, that whereas very few giant cells were present in the cultures infected immediately after feeding with complete medium, a large number of these cells were present in the groups infected 8-12 hours after this time. Both giant and round basophilic cells were extremely rare in the
FIG. 2. Effect of infecting cells at various times after preconditioning by maintenance foi 24 hours in Eagle’s medium without serum, glucose, or glutamine on morphologiral response to infection by Rous sarcoma virus. (A) Preconditioned cells which were not infected with virus, stained 72 hours after refeeding. (B) Cells which had not been preconditioned and which were infected with virus ?%I2 hours after refeeding. (C) Preconditioned cells infected with virus O-4 hours after maintenance in deficient medium. Cultures were removed for staining 72 hours after infection in this group and in groups B and D. (D) I’reconditioned cells exposed to virus 8-12 hours after maintenance in deficient medium. Infection was with Bryan strain virus at an input multiplicity of 1 PFIJ per cell. Cells were third passage turkey fibroblasts which had covered approximately one-half of the glass surface when exposed to the preconditioning procedure. Cells were grown on coverslips and stained with Map-Griinwald Giemsn stain after fixation. Magnification: X 315.
529
530
PRINCE
control groups which had not been maintained in deficient medium. It should be emphasized that the sequence of morphologic responses with time was not consistently reproducible. Significant differences in morphologic response were, however, always observed in experiments in which cells were infected at different times after “preconditioning” by combined low temperature-deficient medium treatment. These experiments provide a further example of the fact that the occurrence and nature of morphological transformation depends on the physiological state of the cells at the time of infection. The observation that morphologic transformation can be varied at will by infecting at time intervals TABLE
2
RESULTS OF STUDY OF SINGLE-FOCUS Virus Bird no.
1 ‘umor no.*
1
-
(Log
IA
1.7
1B
1.7
2
2A
2.0
3
3A
3.82
3B
6.3
3C
6.5
4A
6.2
4B
5.6
4c
K.D.
4
Morphology
ccmtent P FU/g: -
TUMORS~
) _-
Grossc
1Microscopic -
1.5 X 1.5 cm Fleshy 1.5 X 1.5 cm Fleshy 0.6 X 0.3 cm Gelatinous 0.8 X 0.2 cm Gelatinous 1.0 X 0.7 cm Fleshy 0.5 X 0.5 cm Fleshy 1.0 X 0.8 cm Fleshy, firm 1.0 X 1.0 cm Fleshy, hard 1.0 X 0.6 cm Gelatinous
Spindle
cell
Spindle
cell
Giant
cell
Round
cell
Spindle
cell
Spindle
cell
Fusiform cell Spindle
cell
Round
cell
a Six chickens were inoculated intradermally in twelve sites with 10e6.3 dilution of standard virus. Tumors appeared in 9/72 inoculated sites. Another yroup of chickens inoculated with 10m6.0 dilution at the same time developed tumors in 17/72 inoculated sites. h’l’umors were harve&d for assay 16 days after virus inoculation. c Dimension is average diameter times thickness. All tumors were approximately spherical in outline when seen from the skin surface.
after preconditioning offers a suitable system for the analysis of the nature of the biochemical conditions which favor morphological transformation. Morphological Variation in Tumors Produced in Vivo by Single Virus Particles In the course of experiments designed to determine the comparative morphology of virus releasing and “noninfective” (Prince, 1958) tumors, it was observed that marked variation exists in the morphology of tumors induced by single virus particles in viva, irrespective of virus content. Table 2 summarizes the results of one such experiment. Standard strain Rous sarcoma virus was inoculated intradermally into twelve sites on each of six chickens, at a dose known from previous assays to give rise to tumors in only the small fraction of the inoculated sites. Two inoculum potencies were employed: 10W6.0dilution of virus stock, which gave rise to seventeen tumors out of seventy-two sites inoculated; and 10-6.3 dilution of virus stock, which gave rise to nine tumors out of seventy-two sites inoculated. Tumors were harvested for assay and histological examination 16 days after virus inoculation only from the group inoculated with the highest virus dilution. The conclusion that these tumors are derived from the infection of single cells by single virus particles is strongly supported by the finding of a linear relation between the number of small spherical nodules per inoculation site, and dilution of virus inoculated (Prince, 1959a). This conclusion is also supported by the fact that these tumors are invariably circular in outline when seen through the skin, whereas irregularly shaped and dumbbell-shaped massesare seen when t’he inoculum potency is increased. As summarized in Table 2, and illustrated in Fig. 3, four morphological tumor types were observed out of nine tumors examined in this experiment. Five out of the nine tumors examined were of the classical spindlecell fibromyxosarcoma type usually arising as a result of tissue transplantation, or inoculation of larger doses, of RSV (Fig. 3A). Two out of the nine tumors were composed largely of basophilic small round
DETERMINATION
OF CELLULAR
MORPHOLOGY
FIG. 3. Morphology of tumors induced by single virus particles inoculated intradermally into IO-da.y-old chicks. The four morphological types which were observed are illustrated: (9) spindle-ccl1 type fibromysosarcoma; (B) tumor composed entirely of large dcgeneratp giant, cells surrounded by numerous inflammatory cells and an abundant mutinous ground substance; (C) tumor composed largely of small basophilic round ~11s; (II) tumor composed of cells of intermediate morphology varying between round and spintllc in shape. The majority of the cells arc of fusiform shape. -411 tumors were harvested 16 days after virus inoculation from an experiment in which nine out of seventy-two inoculated sites tlavclo~~tl tumors. Tissues were fixed in Bouin’s fluid and stained with hematoxylin and rosin. Magnification: X 790.
531
PRINCE
532
cells illustrated in Fig. 3C. The resemblance of these cells to those comprising the roundcell type of focus will be noted. Both of these in vivo Limorphotypes” were associated with both tumors of the virus producing and “noninfective” varieties. One out of the nine tumors was composed of fusiform cells intermediate in morphology between round and spindle-cell types. This is illustrated in Fig. 3D. The possibility t’hat these cells correspond to the foci of fusiform cells described by Temin (1960) should be considered. One out of the nine tumors was histologically most unusual in that it was solely composed of somewhat degenerateappearing giant cells (Fig. 3B). Large numbers of inflammatory cells, as well as abundant mutinous stroma, surrounded these cells. The possibility should again be considered that these tumors correspond to the giant-cell foci described by Temin. It should be emphasized that the morphologic appearance of these tumors, particularly those of giant- and round-cell types, was quite uniform in the sections examined. The tumors were, however, small (about 1.0 cm, seeTable 2) at the time of examination. It is thus possible that the uniformity of appearance might disappear with time and growth in the animal. The possible significance of these alternate in vivo responsesto infect,ion with Rous sarcoma virus will be discussed further below. DISCUSSION
It is now clear from the results of this and other studies that Rous-infected, virus-producing cells need not be morphologically transformed. The presently reported instances of this phenomenon offer useful opportunities for further analysis into mechanism. For instance, it would be of interest to determine the active components in tryptose phosphate broth which facilitate morphological t.ransformation. Similarly, a detailed biochemical analysis of the nature of the probable partial synchronization of synthetic processeswhich follows upon the 24hour period of starvation and exposure to low temperature employed in the preconditioning experiments herein reported, may
offer important clues into the requirements for transformation on a molecular level. Of crucial importance to an understanding of these phenomena, as well as to an understanding of the mechanism of tumorigenesis by Rous sarcoma virus, is the question whether nontransformed virus-producing cells are actually neoplastic. The fact that there is a ratio of lo:20 between the number of infectious virus particles (in the sense of particles which are able to convert cells into virus yielders) and number of particles det.ected by their ability to transform cells morphologically in either focus assay or chorioallantoic membrane pock assay ; and that the latter two assays are identical in sensitivity to assays which depend on the ability of virus to give rise to tumors in sensitive animals (Prince, 1958, 1960a; Rubin, 1960; Gold& and Vigier, 1961) would perhaps be most compatible with the interpretation that only transformed cells are neoplast,ic. The fact that Rous sarcoma virus can apparently multiply within a cell without inducing morphological alteration is also in accord with the findings of Temin (1961) in regard to dual infection of cells by virus of differing genotype. The genetic markers employed in these experiments were those governing the nature of morphological transformation. It was found that cells could be concurrently infected by virus of two genotypes, and that progeny of such cells could continue to release virus of both genotypes for long periods of time. Of great interest, however, was the finding that only one genotype could control morphology, and that this could be either that of the original or superinfecting virus. These observations obviously lend themselves to the hypothesis that there is a cert,ain site into which the virus genome must integrate for it to be able to express it,s morphologic, and perhaps also tumorigenic, potentialities. The ratio of IO:20 between the ratio of infectious particles and the number of morphologically converting, or tumor-forming, particles can be explained by assuming that there is a probability of only 1 in lo-20 that the infecting viral genome can reach the critical site (nuclear Yntegration” site?)
DETERMINATION
OF CELLULAR
required for complete expression of its potentialities. A puzzling corollary of this hypothesis would be the question why a nonconverted virus-yielding cell does not produce tumors in viva by infection and conversion of neighboring cells. The following hypothesis could be raised: The conversion process is apparently inefficient (1 in 10-20, see above) ; thus, conversion of neighboring cells may be a slow, and even a relatively rare event (the role of interferon-like substances in inhibition of infection of neighboring cells might be considered in this regard). Evidence that this is so may be adduced from the observation that virus-containing, and noninfective (non-virus-containing), single-focus tumors do not vary significantly in size (Prince, 1959b) ; and that size and growth rate of virus-containing t,umors is not significantly affected by presence of antibody in host animals (Vigier, 1958). Late-appearing transformed cells may well be inhibited from displaying their neoplastic potential by immune reactivity of host lymphocytes against new antigens in transformed cells. The finding of four morphologically distinct tumor types out of nine tumors arising from infection by single virus particles can obviously be explained in at least three different ways. First, one must consider the possibility that t,hese tumors arose by infection of four genetically different virus particles. Such a hypothesis would appear a priori unlikely since such a frequency of mutants has never been described as occurring in an unselected population. It is also noteworthy in this connection that Temin’s mutant strains which gave rise t,o foci of different morphotypes in vitro produced indist,inguishable tumors in vivo. It would be of great interest to know whether this would also occur if tumors were induced by single virus particles of these genetically marked st.rains. A second hypothesis which must be considered is that the different tumors arose as a result of infection of cells of different types. Indeed, in Temin’s study of determination of morphology in vitro an effect of cell type on morphology was observed. This
MORPHOLOGY
533
hypothesis could, therefore, account for some or all of the observed variation, The third hypothesis which must be considered is that the morphologic variation depends on the physiologic state of the cells at time of infection. This hypothesis would be in accord with the observations described in this report, namely, that different proportions of basophilic round cells and giant cells were seen, depending on the time of infection of cells after preconditioning by starvation. The data presently at hand do not rule out the hypothesis that the morphologically unconverted cell in vitro, seen in media without tryptose phosphate broth, and in cells maint,ained in the presence of high concent.rations of fetal calf sera (Rubin, 1960), and under other circumst,ances, may correspond t.o the cell observed in viva as a spindle cell. It is of prime importance to determine whether morphological conversion and the acquisit.ion of neoplastic properties are necessarily associated. If such an association can be experimentally demonst’rated, the underst,anding of the mechanism of the phenomena described in this report will become crucial to an understanding of the mechanism of carcinogenicit,y of ROW sarcoma virus. ACKNOWLEDGMENTS The McGill edged.
capable technical assistance of Mrs. Mary and Miss Joan Ferris is gratefully acknowl-
REFEREKCES A., and VIGIER, P. (1961). Growth of Rous sarcoma virus and cells in confluent chick embryo monolayers. Virology 15, 3646. HALBERSTAEDTER, I,., DOLJAAXKI, L., and TENENBAUY, E. (1941). Esperiment~s on the cancerization of cells in vitro by means of Rous sarcoma agent. Brit. J. Etptl. Pathol. 22, 179-187. MA~YAKER, R. A., and GROUP& V. (1956). Discrete foci of altered chicken embryo cells associated with Rous sarcoma virus in tissue culture. Virol-
GOLD&
ogy 2,839~840. A. M. (1958). Rous sarcoma virus. sarcoma virus on the of the chick embryo. 147-159.
PRISCE,
Quantitative studies on I. The titration of Rous chorio-allantoic membrane J. Natl. Cancer Inst. 20,
534
PRINCE
PRINCE, A. M. (1959~~). Studies on the mechanism of induction of neoplasia by Rous sarcoma virus. Acta Unio Intern. contra Cancrum 15, 832-836. PRINCE, A. M. (1959b). Quantitative studies on Rous sarcoma virus. IV. An investigation of the nature of “noninfective” tumors induced by low doses of virus. J. Natl. Cancer Inst. 23, 1361-1381. PRINCE, A. M. (1960a). Quantitative studies on Rous sarcoma virus. V. An analysis of the mechanism of virulence of the Bryan “high titer” strain of RSV. Virology 11, 371399. PRINCE, A. M. (1960b). Quantitative studies on Rous sarcoma virus. VI. Clonal analysis of in vitro infections. Virology 11, 400-424.
RUBIN, H. (1960). The suppression of morphological alterations in cells infected with Rous sarcoma virus. Virology 12, 14-31. TEMIN, H. M. (1960). The control of cellular morphology in embryonic cells infected with Rous sarcoma virus in vitro. Virology 10, I%?-
197. TEWN, H. M. (1961). Mixed infection with two types of ROW sarcoma virus. Virology 13, 158163. VIGIER, P. (1958). Recherches quantitatives sur le sarcome de Rous. Croissances de sarcomes dermiques et anticorps neutralisants. Bull. USsot. franC. &de cancer 45, 460-475.
18,
Factors
524-534 (1962)
Influencing in Cells
the
Determination
Infected
with
ALFRED Lkpartment
of Pathology, New
Rous
Sarcoma
Morphology Virus’
M. PRINCE2
Yale Haven,
Accepted
of Cellular
University Connecticut July
School
of Medicine,
23, 1962
Additional means have been described for controlling the presence or absence, and nature, of morphological transformation induced by Rous sarcoma virus infection of turkey fibroblasts in vitro. Eagle’s medium supplemented with serum from various species was found to support only limited morphological transformation under normal circumstances. The addition of 10% tryptose broth to such media markedly favored the production of morphological transformation. Infection of cells at various times after a 24-hour period of starvation in serum-free Eagle’s solution without glucose and glutamine at room temperature resulted in marked variations in both the total extent of morphological transformation and the nature of the transformation which was observed. Tumors induced in viva by single virus particles varied widely in morphology. Tumors induced in this manner having predominantly spindle-cell, giant-cell, fusiformcell, and round-cell morphologies were observed. The above findings are discussed particularly in reference to possible correlations between in vitro and in viva morphology, and in vitro morphological conversion with neoplastic potential; and possible mechanisms underlying the production of morphologic conversion by Rous sarcoma virus. INTRODUCTIOK
It is a unique property of the Rous sarcoma virus that cells maintained in vitro may be rapidly converted by infection into cells having various types of altered morphology. The most common alteration is the appearance of the round basophilic cells first described by Halberst,aedter et al. (1941). The conversion of cells to this type of morphology was shown by Manaker and Groupi! (1956) to provide a means for in vitro assay of Rous sarcoma virus, since under suitable condtions these cells proliferated into discrete countable foci. l These studies were supported by research grants from the American Cancer Society, and from the Jane Coffin Childs Fund for Medical Research. ‘Present address: Wistar Institute, Philadelphia, Pennsylvania.
In 1960 Temin report,ed additional types of morphological conversion and analyzed some of the factors determining the production of alternate morphological responses. It was found that virus derived from certain clones had the property of converting cells to long spindle-shaped morphology; and that this ability reflected a genetic property of the virus. On the other hand, isolation of virus clones from foci having giant-cell or fusiform-cell morphology revealed that this response did not depend on the genetic nature of the infecting virus, since these clones produced primarily round-cell foci. It appeared that the production of giantcell and fusiform alteration depended on the physiological state of the cells at the time of infection. Temin (1960) also has reported that RSV-infected cells may undergo no mor-
524
DETERMINATION
OF CELLULAR
phological alteration. This was observed when infected monolayers were not fed at regular intervals and was also observed in the infection of a cloned line of fibroblasts which had been maintained for 3 months in vitro prior to infection. With these cells the number of foci of morphologically transformed cells was far below the number of cells shown to have been infected by infectious center assays. That not all virus-infected cells show morphological transformation may also be concluded from the finding that in certain tissue culture media only a small fraction of RSV-producing cell clones can be morphologically distinguished from uninfected control clones (Prince, 1960b) ; and from the finding that fetal calf serum can suppressthe appearance of foci of morphologically altered cells following infection of monolayers with small amounts of RSV, but does not change the proportion of infected cells (Rubin, 1960). The above studies raise the important and so far inadequately answered question as to what correlation exists between in vitro morphology and in viva behavior. It, is of interest, in this regard that inoculation of virus stocks having genetic markers for round-cell or spindle-cell conversion resulted in t’umors which were morphologically indistinguishable (personal communication, I-I. M. Temin). The present, report will present evidence that the morphological responsecan be controlled in vitro by choice of tissue culture media, and by infecting cells at various times after preconditioning. In addition, it will be shown that tumors produced in viva by single virus particles may vary significantly in morphology and that the morphological variations observed in viva appear to correspond t,o those which have been dcscribed to occur in vitro.
MORPHOLOGY
525
assay was by the chorioallantoic membrane technique (Prince, 1958). Histological Procedures Tissue cult,ures for morphological examination were grown on coverslips in Leighton tubes. Coverslips were fixed in absolute met.hanol and stained by the MayGriinwald Giemsa procedure. Tumor tissues were fixed in Bouin’s solution and stained with hematoxylin and eosin. Procedure for Infection of Cultures at Various Times after Preconditioning
Turkey fibroblests cultivated in Eagle’s medium containing 5% calf and 5% turkey serum were permitted to grow unt,il approximately one-half of the surface of the glass had been covered. At this time cultures were washed with “deficient medium” (Eagle’s solution lacking serum, glucose, and glutamine). A proportion of the cultures were then fed with t.he “deficient medium” and held for 24 hours at room temperature. A second group of cultures was fed with complete growth medium and held for 24 hours at 37°C. At the end of t,he 24-hour preconditioning period, a group of cultures was immediately infected by addition of approximately 1 plaque-forming unit (PFU) per cell of Bryan strain Rous sarcoma virus suspended in 0.2 ml of complete medium. The remaining cultures were fed with complete medium. After 5 additional hours at 37”, infected cult,ures were washed four t,imes with PBS and refed with complete medium. At t,his time a second group of cultures was infected as above and allowed to remain in the presenceof the virus inoculum for a 5-hour period. Addit,ional cultures were infected for the periods 10-15, 15-20, and 20-25 hours following preconditioning. Similar groups of cultures were exposed to inocuia lacking virus but were otherwise MSTERIALS AND METHODS treated as described above. All cultures were Virus strains, and tissue culture proce- fed with complete growth medium 48 hours dures, employed in this study have been after termination of the preconditioning previously described (Prince, 1960a). The period. Cultures were stained for histological exchickens employed were from the Cornucopia (“Pure Hope”) line of White Leghorns amination, and supernates removed for virus asSay, 72 or 96 hours after infection. previously described (Prince, 1958). Virus
PRINCE
526
ever, characteristic basophilic round cells were extremely rare. Similar results were Effect of Maintenance Medium on Morphoobtained with Eagle’s media containing all logical Responseof Turkey Fibroblasts the serum types and combinations described to Infection by RSV in Vitro above when tryptose phosphate broth was It was a puzzling fact that in the course not present. In contrast to these results, of three years of study of various aspects cultures overlaid wit,h tryptose phosphate broth containing medium showed conversion of the infection of chick and turkey fibroof the majority of the cells in the culture blasts by RSV in vitro, morphological transto intensely basophilic round cells (Fig. formation of the type observed by Manaker and Group&, and Rubin and Temin, had 1B). Assays carried out on supernatant fluids never been observed. This was the case even with Bryan strain virus, with which it could from sister cultures to those portrayed in be shown that the majority of the cells in Fig. 1, taken 70 hours after infection, rethe culture rapidly became converted into vealed 106.6 PFU/ml in cultures overlaid virus producers (Prince, 1960a). The media with ET&, and 105.0PFU/ml in cultures employed in these st,udies (in which mor- overlaid wit,h the tryptose phosphate broth phological conversion did not occur) were containing medium. There were thus cerEagle’s basal medium containing either tainly no fewer virus producing cells in 10% duck serum (ED,,), 10% turkey serum cultures overlaid with media lacking tryp(ET,,), 10% calf serum (ECi,) , 5% duck tose phosphate, in which morphological serum plus 5% calf serum (ED&), or 5% transformation did not occur. turkey serum plus 5% calf serum (ET&s). It appeared likely that the absence of mor- Effect of Preconditioning on Morphological Response of Fibroblasts to Infection by phological conversion was a consequence of Rous Sarcoma Virus the difference in the media employed in these studies as compared to the media emSeveral experiments were carried out to ployed in studies in which morphological determine the effect of infecting fibroblasts conversion was observed. The most obvious at various time int.ervals after preconditiondifference between such media was the ing in an attempt to produce a degree of association of tryptose phosphate broth con- biochemical synchrony. Table 1 summarizes taining media with the presence of morpho- the results of one such experiment in which logical conversion. tenth passage turkey fibroblasts were inExperiments were therefore carried out fected at an input multiplicity of 1 PFU per in which turkey fibroblasts were infected cell with Bryan strain virus at various times with Bryan strain virus at an input mult,iafter being held for 24 hours at room templicity of 10 PFU per cell. Cultures were perature in a deficient medium composed washed twice and overlaid with different of Eagle’s solution lacking serum, glucose, media after being held for 2 hours at 37” to and glutamine. It will be noted that this permit, virus adsorption. The result,s of an procedure did not significantly affect the experiment in which cultures were overlaid yield of virus in the supernate 4 days after with Eagle’s medium containing 5% calf infection. Other experiments, in which inand 5% turkey serum or with EagIe’s me- fectious center assays were done, similarly dium containing 10% duck serum and 10% failed to reveal significant variations. Howtryptose phosphate broth are presented in ever, microscopic examination of these culFig. 1. Infected cultures overlaid with me- tures 4 days after infection revealed that dium lacking tryptose phosphate broth the preconditioning procedure did have a were indistinguishable from cont,rols when marked effect on the degree of morphologiexamined without staining, 51 hours after cal transformation. Whereas control culinfection. In stained preparations there was tures, which had not been maintained in the noticeable increase in cytoplasmic baso- deficient medium, showed no significant philia in infected cultures (Fig. ID) ; how- degree of morphological transformation. RESULTS
DETERMINATION
OF CELLULAR
MORPHOLOGY
FIG. 1. Effect of maintenance medium on morphologic conversion of turkey fibroblasts b: Rous sarcoma virus (Bryan strain). (A) and (B) show turkey fibroblasts maintained in Eagle’s medium with 10% duck serum and 10% tryptose phosphate broth. Control uninfected cells are shown in (a); cells infected 51 hours previously in (B). (C) and (D) show turke\ fibroblasts maintained in Eagle’s medium wit.h 5% turkey and 5% calf serum. (C) shows control uninfected cells, and (D), cells infected with virus 51 hours previously. Magnification: x 315.
527
PRINCE
528
TABLE EFFECT
OF PRECONDITIONING INFECTION Preconditioning
prior
ON MORPHOLOGICAL BY RSV (BRYAN
‘emp. (“C)
RESPONSE “HIGH TITER”
-
to infection”
1st Incubation Medium
1
4 Days
2nd Incubation
-
rime (hr)
Medium -
Complete” Complete Complete Complete Complete
37 37 37 37 37
24 24 24 24 24
Complete Complete Complete Complete
Deficient* Deficient Deficient Deficient Deficient
21 21 21 21 21
24 24 24 24 24
Complete Complete Complete Complete
T ‘emp. w 37 37 37 37
-
Time (h-1 -
-I
after
PFU in supernate per cell in culture
FIBROBLASTS
infection
I
EEstimated yO ,:ells “transformed”d
I-
TO
Estimated “coverage” lIdday after transfer8
-
5 10 15 20
7.1 4.6 2.8 4.1 3.6
5 10 15 20
3.4 2.8 7.0 4.1 5.4
10
37 37 37 37
OF TURKEY STRAIN)
a Complete medium = Eagle’s with 5y0 calf and 57, duck serum. * Deficient medium = Eagle’s solution without serum, glucose, or glutamine. c Cultures were infected with 1 PFU/cell (input), washed for four times after a 5-hour adsorption, period, and refed complete medium. Cells were tenth passage turkey fibroblasts. “epithelioid” (fusiform) or giant morphology. d Transformed cells = cells having e Cultures trypsinized and subcultured 1:4 on fourth day after infection. “Coverage” refers to proportion of glass-surface covered by cells.
there was significant transformation to fusiform “epithelioid,” or giant cells, in cultures infected immediately after maintenance in deficient medium. Cultures infected between 5 and IO hours after refeeding following maintenance in deficient media resembled control cultures in that morphological transformation was minimal or absent. An especially marked degree of morphological transformation was noted in cultures infected between 15 and 25 hours after refeeding following maintenance in deficient medium. In this experiment cultures in each group were trypsinized 4 days after infection and divided 1 to 4 for subcultivation. When subcultures were examined 1 day after transfer, marked differences were readily noted in the degree of coverage of the glass surface by the subcultured cells. These differences corresponded to the degree of transformation previously observed. A large number of round cells were seen floating in the medium of the subcultures from the transformed
cultures. It is likely, since rounded cells are known to attach poorly to glass surfaces, that these cells were viable but differed from the untransformed cells in their ability to attach to the glass surface. The morphology of cells from an experiment which differed from the above only in that cultures were infected at 4-hour intervals following refeeding after maintenance in deficient medium, and that third passage turkey fibroblasts were employed, are presented in Fig. 2. Cells were examined 72 hours after infection. In this experiment the most marked degree of transformat,ion to basophilic round cells occurred in the cultures infected immediately after maintenance in deficient medium. It was observed, however, that whereas very few giant cells were present in the cultures infected immediately after feeding with complete medium, a large number of these cells were present in the groups infected 8-12 hours after this time. Both giant and round basophilic cells were extremely rare in the
FIG. 2. Effect of infecting cells at various times after preconditioning by maintenance foi 24 hours in Eagle’s medium without serum, glucose, or glutamine on morphologiral response to infection by Rous sarcoma virus. (A) Preconditioned cells which were not infected with virus, stained 72 hours after refeeding. (B) Cells which had not been preconditioned and which were infected with virus ?%I2 hours after refeeding. (C) Preconditioned cells infected with virus O-4 hours after maintenance in deficient medium. Cultures were removed for staining 72 hours after infection in this group and in groups B and D. (D) I’reconditioned cells exposed to virus 8-12 hours after maintenance in deficient medium. Infection was with Bryan strain virus at an input multiplicity of 1 PFIJ per cell. Cells were third passage turkey fibroblasts which had covered approximately one-half of the glass surface when exposed to the preconditioning procedure. Cells were grown on coverslips and stained with Map-Griinwald Giemsn stain after fixation. Magnification: X 315.
529
530
PRINCE
control groups which had not been maintained in deficient medium. It should be emphasized that the sequence of morphologic responses with time was not consistently reproducible. Significant differences in morphologic response were, however, always observed in experiments in which cells were infected at different times after “preconditioning” by combined low temperature-deficient medium treatment. These experiments provide a further example of the fact that the occurrence and nature of morphological transformation depends on the physiological state of the cells at the time of infection. The observation that morphologic transformation can be varied at will by infecting at time intervals TABLE
2
RESULTS OF STUDY OF SINGLE-FOCUS Virus Bird no.
1 ‘umor no.*
1
-
(Log
IA
1.7
1B
1.7
2
2A
2.0
3
3A
3.82
3B
6.3
3C
6.5
4A
6.2
4B
5.6
4c
K.D.
4
Morphology
ccmtent P FU/g: -
TUMORS~
) _-
Grossc
1Microscopic -
1.5 X 1.5 cm Fleshy 1.5 X 1.5 cm Fleshy 0.6 X 0.3 cm Gelatinous 0.8 X 0.2 cm Gelatinous 1.0 X 0.7 cm Fleshy 0.5 X 0.5 cm Fleshy 1.0 X 0.8 cm Fleshy, firm 1.0 X 1.0 cm Fleshy, hard 1.0 X 0.6 cm Gelatinous
Spindle
cell
Spindle
cell
Giant
cell
Round
cell
Spindle
cell
Spindle
cell
Fusiform cell Spindle
cell
Round
cell
a Six chickens were inoculated intradermally in twelve sites with 10e6.3 dilution of standard virus. Tumors appeared in 9/72 inoculated sites. Another yroup of chickens inoculated with 10m6.0 dilution at the same time developed tumors in 17/72 inoculated sites. h’l’umors were harve&d for assay 16 days after virus inoculation. c Dimension is average diameter times thickness. All tumors were approximately spherical in outline when seen from the skin surface.
after preconditioning offers a suitable system for the analysis of the nature of the biochemical conditions which favor morphological transformation. Morphological Variation in Tumors Produced in Vivo by Single Virus Particles In the course of experiments designed to determine the comparative morphology of virus releasing and “noninfective” (Prince, 1958) tumors, it was observed that marked variation exists in the morphology of tumors induced by single virus particles in viva, irrespective of virus content. Table 2 summarizes the results of one such experiment. Standard strain Rous sarcoma virus was inoculated intradermally into twelve sites on each of six chickens, at a dose known from previous assays to give rise to tumors in only the small fraction of the inoculated sites. Two inoculum potencies were employed: 10W6.0dilution of virus stock, which gave rise to seventeen tumors out of seventy-two sites inoculated; and 10-6.3 dilution of virus stock, which gave rise to nine tumors out of seventy-two sites inoculated. Tumors were harvested for assay and histological examination 16 days after virus inoculation only from the group inoculated with the highest virus dilution. The conclusion that these tumors are derived from the infection of single cells by single virus particles is strongly supported by the finding of a linear relation between the number of small spherical nodules per inoculation site, and dilution of virus inoculated (Prince, 1959a). This conclusion is also supported by the fact that these tumors are invariably circular in outline when seen through the skin, whereas irregularly shaped and dumbbell-shaped massesare seen when t’he inoculum potency is increased. As summarized in Table 2, and illustrated in Fig. 3, four morphological tumor types were observed out of nine tumors examined in this experiment. Five out of the nine tumors examined were of the classical spindlecell fibromyxosarcoma type usually arising as a result of tissue transplantation, or inoculation of larger doses, of RSV (Fig. 3A). Two out of the nine tumors were composed largely of basophilic small round
DETERMINATION
OF CELLULAR
MORPHOLOGY
FIG. 3. Morphology of tumors induced by single virus particles inoculated intradermally into IO-da.y-old chicks. The four morphological types which were observed are illustrated: (9) spindle-ccl1 type fibromysosarcoma; (B) tumor composed entirely of large dcgeneratp giant, cells surrounded by numerous inflammatory cells and an abundant mutinous ground substance; (C) tumor composed largely of small basophilic round ~11s; (II) tumor composed of cells of intermediate morphology varying between round and spintllc in shape. The majority of the cells arc of fusiform shape. -411 tumors were harvested 16 days after virus inoculation from an experiment in which nine out of seventy-two inoculated sites tlavclo~~tl tumors. Tissues were fixed in Bouin’s fluid and stained with hematoxylin and rosin. Magnification: X 790.
531
PRINCE
532
cells illustrated in Fig. 3C. The resemblance of these cells to those comprising the roundcell type of focus will be noted. Both of these in vivo Limorphotypes” were associated with both tumors of the virus producing and “noninfective” varieties. One out of the nine tumors was composed of fusiform cells intermediate in morphology between round and spindle-cell types. This is illustrated in Fig. 3D. The possibility t’hat these cells correspond to the foci of fusiform cells described by Temin (1960) should be considered. One out of the nine tumors was histologically most unusual in that it was solely composed of somewhat degenerateappearing giant cells (Fig. 3B). Large numbers of inflammatory cells, as well as abundant mutinous stroma, surrounded these cells. The possibility should again be considered that these tumors correspond to the giant-cell foci described by Temin. It should be emphasized that the morphologic appearance of these tumors, particularly those of giant- and round-cell types, was quite uniform in the sections examined. The tumors were, however, small (about 1.0 cm, seeTable 2) at the time of examination. It is thus possible that the uniformity of appearance might disappear with time and growth in the animal. The possible significance of these alternate in vivo responsesto infect,ion with Rous sarcoma virus will be discussed further below. DISCUSSION
It is now clear from the results of this and other studies that Rous-infected, virus-producing cells need not be morphologically transformed. The presently reported instances of this phenomenon offer useful opportunities for further analysis into mechanism. For instance, it would be of interest to determine the active components in tryptose phosphate broth which facilitate morphological t.ransformation. Similarly, a detailed biochemical analysis of the nature of the probable partial synchronization of synthetic processeswhich follows upon the 24hour period of starvation and exposure to low temperature employed in the preconditioning experiments herein reported, may
offer important clues into the requirements for transformation on a molecular level. Of crucial importance to an understanding of these phenomena, as well as to an understanding of the mechanism of tumorigenesis by Rous sarcoma virus, is the question whether nontransformed virus-producing cells are actually neoplastic. The fact that there is a ratio of lo:20 between the number of infectious virus particles (in the sense of particles which are able to convert cells into virus yielders) and number of particles det.ected by their ability to transform cells morphologically in either focus assay or chorioallantoic membrane pock assay ; and that the latter two assays are identical in sensitivity to assays which depend on the ability of virus to give rise to tumors in sensitive animals (Prince, 1958, 1960a; Rubin, 1960; Gold& and Vigier, 1961) would perhaps be most compatible with the interpretation that only transformed cells are neoplast,ic. The fact that Rous sarcoma virus can apparently multiply within a cell without inducing morphological alteration is also in accord with the findings of Temin (1961) in regard to dual infection of cells by virus of differing genotype. The genetic markers employed in these experiments were those governing the nature of morphological transformation. It was found that cells could be concurrently infected by virus of two genotypes, and that progeny of such cells could continue to release virus of both genotypes for long periods of time. Of great interest, however, was the finding that only one genotype could control morphology, and that this could be either that of the original or superinfecting virus. These observations obviously lend themselves to the hypothesis that there is a cert,ain site into which the virus genome must integrate for it to be able to express it,s morphologic, and perhaps also tumorigenic, potentialities. The ratio of IO:20 between the ratio of infectious particles and the number of morphologically converting, or tumor-forming, particles can be explained by assuming that there is a probability of only 1 in lo-20 that the infecting viral genome can reach the critical site (nuclear Yntegration” site?)
DETERMINATION
OF CELLULAR
required for complete expression of its potentialities. A puzzling corollary of this hypothesis would be the question why a nonconverted virus-yielding cell does not produce tumors in viva by infection and conversion of neighboring cells. The following hypothesis could be raised: The conversion process is apparently inefficient (1 in 10-20, see above) ; thus, conversion of neighboring cells may be a slow, and even a relatively rare event (the role of interferon-like substances in inhibition of infection of neighboring cells might be considered in this regard). Evidence that this is so may be adduced from the observation that virus-containing, and noninfective (non-virus-containing), single-focus tumors do not vary significantly in size (Prince, 1959b) ; and that size and growth rate of virus-containing t,umors is not significantly affected by presence of antibody in host animals (Vigier, 1958). Late-appearing transformed cells may well be inhibited from displaying their neoplastic potential by immune reactivity of host lymphocytes against new antigens in transformed cells. The finding of four morphologically distinct tumor types out of nine tumors arising from infection by single virus particles can obviously be explained in at least three different ways. First, one must consider the possibility that t,hese tumors arose by infection of four genetically different virus particles. Such a hypothesis would appear a priori unlikely since such a frequency of mutants has never been described as occurring in an unselected population. It is also noteworthy in this connection that Temin’s mutant strains which gave rise t,o foci of different morphotypes in vitro produced indist,inguishable tumors in vivo. It would be of great interest to know whether this would also occur if tumors were induced by single virus particles of these genetically marked st.rains. A second hypothesis which must be considered is that the different tumors arose as a result of infection of cells of different types. Indeed, in Temin’s study of determination of morphology in vitro an effect of cell type on morphology was observed. This
MORPHOLOGY
533
hypothesis could, therefore, account for some or all of the observed variation, The third hypothesis which must be considered is that the morphologic variation depends on the physiologic state of the cells at time of infection. This hypothesis would be in accord with the observations described in this report, namely, that different proportions of basophilic round cells and giant cells were seen, depending on the time of infection of cells after preconditioning by starvation. The data presently at hand do not rule out the hypothesis that the morphologically unconverted cell in vitro, seen in media without tryptose phosphate broth, and in cells maint,ained in the presence of high concent.rations of fetal calf sera (Rubin, 1960), and under other circumst,ances, may correspond t.o the cell observed in viva as a spindle cell. It is of prime importance to determine whether morphological conversion and the acquisit.ion of neoplastic properties are necessarily associated. If such an association can be experimentally demonst’rated, the underst,anding of the mechanism of the phenomena described in this report will become crucial to an understanding of the mechanism of carcinogenicit,y of ROW sarcoma virus. ACKNOWLEDGMENTS The McGill edged.
capable technical assistance of Mrs. Mary and Miss Joan Ferris is gratefully acknowl-
REFEREKCES A., and VIGIER, P. (1961). Growth of Rous sarcoma virus and cells in confluent chick embryo monolayers. Virology 15, 3646. HALBERSTAEDTER, I,., DOLJAAXKI, L., and TENENBAUY, E. (1941). Esperiment~s on the cancerization of cells in vitro by means of Rous sarcoma agent. Brit. J. Etptl. Pathol. 22, 179-187. MA~YAKER, R. A., and GROUP& V. (1956). Discrete foci of altered chicken embryo cells associated with Rous sarcoma virus in tissue culture. Virol-
GOLD&
ogy 2,839~840. A. M. (1958). Rous sarcoma virus. sarcoma virus on the of the chick embryo. 147-159.
PRISCE,
Quantitative studies on I. The titration of Rous chorio-allantoic membrane J. Natl. Cancer Inst. 20,
534
PRINCE
PRINCE, A. M. (1959~~). Studies on the mechanism of induction of neoplasia by Rous sarcoma virus. Acta Unio Intern. contra Cancrum 15, 832-836. PRINCE, A. M. (1959b). Quantitative studies on Rous sarcoma virus. IV. An investigation of the nature of “noninfective” tumors induced by low doses of virus. J. Natl. Cancer Inst. 23, 1361-1381. PRINCE, A. M. (1960a). Quantitative studies on Rous sarcoma virus. V. An analysis of the mechanism of virulence of the Bryan “high titer” strain of RSV. Virology 11, 371399. PRINCE, A. M. (1960b). Quantitative studies on Rous sarcoma virus. VI. Clonal analysis of in vitro infections. Virology 11, 400-424.
RUBIN, H. (1960). The suppression of morphological alterations in cells infected with Rous sarcoma virus. Virology 12, 14-31. TEMIN, H. M. (1960). The control of cellular morphology in embryonic cells infected with Rous sarcoma virus in vitro. Virology 10, I%?-
197. TEWN, H. M. (1961). Mixed infection with two types of ROW sarcoma virus. Virology 13, 158163. VIGIER, P. (1958). Recherches quantitatives sur le sarcome de Rous. Croissances de sarcomes dermiques et anticorps neutralisants. Bull. USsot. franC. &de cancer 45, 460-475.