Nov 22, 1972 - Department of Pathology, New York University School of Medicine, New York, New York ... We have also isolated new tsSV3T3 lines, using cells which had been infected ..... and 39 C. However, more recently Holley and.
JouRNAL OF VIROLOGY, May 1973. p. 702-708 Copyright 0 1973 American Society for Microbiology
Vol. 11, No. 5 Printed in U.S.A.
Temperature-Sensitive Simian Virus 40-Transformed Cells: Phenomena Accompanying Transition from the Transformed to the "Normal" State HARTMUT C. RENGER AND CLAUDIO BASILICO Department of Pathology, New York University School of Medicine, New York, New York 10016
Received for publication 22 November 1972
Temperature-sensitive simian virus (SV 40)-transformed 3T3 cells (tsSV3T3), which express the transformed phenotype when growing at 32 C but not at 39 C, were used to study changes in growth behavior during shift-up or shift-down experiments. In cultures of tsSV3T3 cells which had reached or were beyond monolayer density at 32 C, DNA synthesis reached very low levels within 24 to 48 h after shift-up. When cells which had been allowed to grow to high densities at 32 C were shifted to 39 C, not only cell growth stopped, but within two to three days the cultures shed a large number of cells into the medium. These cells were nonviable, and shedding stopped only when the number of cells attached had been reduced to that characteristic of the saturation density at 39 C. 'l'he remaining attached cells were viable and after the shift to 32 C were again able to grow from the monolayer to high cell densities. This behavior has been compared with that of normal 3T3 and wild-type SV3T3 cells under different conditions. We have also isolated new tsSV3T3 lines, using cells which had been infected with non-mutagenized wild-type SV40. This further demonstrates that the temperature sensitivity of these lines is due to a cellular rather than a viral mutation. It is important to define and clarify the regulation of virally transformed cells, namely, how viral and cellular genes control the expression of malignant transformation. Useful tools for this purpose are transformed cell lines which, because of viral or cellular alterations, express the transformed phenotype in a conditional manner (2, 5, 6, 9). The study of such mutants provides useful insights into the mechanism of cell transformation by viruses. In a previous paper, we reported the isolation of simian virus (SV40)-transformed 3T3 mouse cells which express the transformed phenotype in a temperature-dependent manner (9). These cells, in spite of the fact that they have been isolated from cell populations infected with mutagenized SV40 virus, seem to owe their behavior to a cellular rather than a viral mutation, since, after fusion with permissive monkey cells, wild-type virus is recovered. Four parameters of malignant growth in vitro are temperature sensitive in these cell lines: (i) high saturation density in culture; (ii) ability to form colonies on monolayers of 3T3 cells; (iii) lack of contact inhibition of DNA synthesis; (iv) ag-
glutination by plant lectins such as wheat germ agglutinin and concanavalin A (5a). These phenotypic characteristics are expressed at 32 C, but not at 39 C, and are fully reversible. It was also shown that the tsSV3T3 cells have not irreversibly lost their potential to express the transformed phenotype at 39 C, since infection with murine sarcoma virus renders them fully transformed at any temperature (8). In this paper, we present additional data on the characterization of the ts transformed lines. We have investigated the kinetics of phenotypic reversion after shift-up and the phenomena which accompany this transition. In addition, we have obtained similar ts lines using nonmutagenized SV40 for infection. This supports the conclusion that these mutations are of cellular origin. MATERIALS AND METHODS Cells. The isolation of tsSV3T3 cells has been reported. For the experiments described in this paper, the H6 and H6-15 lines were used (9). Untransformed
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TEMPERATURE-SENSITIVE SV40-TRANSFORMED CELLS
parental 3T3 mouse fibroblasts (11) and wild-type SV3T3 (SV-101) (7) were used as control lines. Media. The cells were generally grown in Dulbecco's modification of Eagle medium supplemented with 5 or 10% calf serum. If high concentrations of serum were used (e.g., 50%), the serum was dialyzed against medium for at least 24 h before use. DNA synthesis. DNA synthesis was determined by incorporation of radioactively labeled thymidine into acid-insoluble material. The cultures were washed twice with isotonic Tris-hydrochloride buffer and lysed in 1% sodium dodecyl sulfate. After 10 min at room temperature, the lysate was scraped off with a rubber policeman. The viscosity was lowered and highmolecular-weight compounds were sheared by strong pipetting. Samples were then taken, precipitated with trichloroacetic acid, filtered on Millipore filters, dried, and counted in a Beckman LS-250 scintillation spectrometer. Autoradiography. Cells were inoculated onto cover slips and incubated at the desired temperature. At the times indicated 3H-TdR (0.5 uCi, 1 Mg/ml) was added. The cells were washed with isotonic buffer 24 h later and fixed in 70% ethanol-acetic acid (9: 1), washed several times in 70% ethanol, and dried. Cover slips were then mounted on microscope slides, covered with nuclear track emulsion (Kodak), dried, and exposed for 3 to 5 days at 4 C before developing and fixing. The cells were stained with hematoxylin. Mitotic index. Cells were incubated with 0.01 ,g of Velban (Grand Island) per ml for 4 h at 39 C, for 6 h at 32 C, fixed in 2% gluteraldehyde for 20 min at 4 C, and stained with Giemsa. Mitotic cells could easily be distinguished from other cells because of their round appearance and greater uptake of the dye. Between 1,000 and 2,000 cells were examined for each mitotic index. T-antigen assays were performed as described (1,
paralleling the decrease of DNA synthesis. In wtSV3T3 lines, both DNA synthesis and mitotic index remain high even at high cell density. The autoradiographic data (Fig. 2) show that the frequency of DNA-synthesizing cells decreases in parallel with the reduction in overall DNA synthesis after the ts transformants reach confluence at 39 C. At 32 C, or in a standard transformant at 39 C, it remains high all the time. These data show conclusively that "contact" inhibition of growth in these cells is paralleled by inhibition of DNA synthesis. This inhibition is slightly retarded as compared to 3T3 cells, but proceeds essentially in an identical fashion. It can be concluded that, with respect to DNA synthesis, these cells behave like transformants at 32 C, but at 39 C their behavior is that of normal, untransformed cells. Reversal of transformation upon shift to the high temperature. To determine how soon after the shift to 39 C the cells revert to a normal state, we performed the following experiment. H6-15 cells growing at 32 C were allowed to reach or surpass the monolayer stage and then shifted to 39 C. Under these conditions, one would expect DNA synthesis to cease in the culture after reversal of transformation is completed, since DNA synthesis in confluent tsSV3T3 cultures at 39 C is very low. The experiment was performed by determining the rate of DNA synthesis in the cultures at 32 C at the time of shift-up and at 39 C at various times
9). RESULTS DNA synthesis in the ts transformants. In a previous publication, we had shown that when the ts transformants were incubated at 32 C, contact inhibition of DNA synthesis was absent or minimal and the cells reached very high densities (9). At 39 C the cells incorporated 3H-thymidine into their DNA at a high rate in sparse culture, but the total incorporation decreased considerably after confluence. We tested whether the reduction of overall DNA synthesis at 39 C was primarily due to a reduction of the number of DNA-synthesizing cells, as would be expected from normal 3T3 cells, or whether it was due to a reduced rate of DNA synthesis in every cell. It was also tested whether a substantial reduction in mitotic index took place after confluence was reached. The data are shown in Fig. 1. It can be seen that the rate of DNA synthesis per culture decreases greatly after confluence is reached in tsH6-15 cells at 39 C, essentially in the same fashion as in normal 3T3 cells. In both cells the mitotic index drops sharply after confluence,
16
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FIG. 1. DNA synthesis and mitotic index in tsSV3T3 cells at 39 C. tsH6-15 (0), wtSV3T3 (0), and 3T3 (A) cells were plated at 39 C at approximately 104 cells/cm2. At the times indicated, DNA synthesis was measured by pulse-labeling the cells for 2 h with '4C-TdR (0.5ACi, 1gg/ml). The mitotic index was determined. In all cultures, confluence was reached at about day 3. Medium was changed on day 5. Left panel, DNA synthesis: radioactivity incorporated into DNA per culture. Right panel, Mitotic index.
RENGER AND BASILICO
704
J. VIROL. A
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FIG. 2. Frequency of cells synthesizing DNA in cultures of tsSV3T3 cells. Twenty-four hours before the times indicated, 3H-TdR was added to the cultures. They were fixed and autoradiography was performed. In all cultures, confluence was reached at about day 3. Medium was changed on the same day. wtSV3T3, 39 C (-O-); 3T3, 39 C (-A-); tsH6-15, 39 C (-*-); tsH6-15, 32 C (-U-).
after the shift by pulsing the cells with '4C-TdR for 1 h. The results are shown in Fig. 3. After an initial increase in the cultures shifted to 39 C (probably due to the higher temperature of incubation), the rate of DNA synthesis declined, reaching a low level at about 24 h after the shift. Thus, with respect to DNA synthesis, reversal of transformation upon shift to 39 C requires about 24 h. The time required for "retransformation" upon shift-down, as already described (9) is about 48 h. Phenomena accompanying the transition from one state to another. When confluent tsSV3T3 cells were shifted from 39 to 32 C, growth resumed, and the cultures attained a high density. When non-confluent cultures of tsSV3T3 cells were shifted from 32 to 39 C, the cells grew confluent and cell growth stopped. However, when the cells were transferred to 39 C after they had already grown beyond confluence, not only cell growth stopped, but the cultures shed a large number of cells into the medium. This phenomenon started about 24 h after the shift and seemed to subside only after the number of cells attached had been reduced to that characteristic of the saturation density at 39 C, i.e., -6 x 104 cells/cm2. As illustrated in Fig. 4, the phenomenon can be demonstrated
FIG. 3. Kinetics of DNA synthesis in the tsSV3T3 after shift-up. tsH6-15 cells, growing at 32 C, were shifted to 39 C, and the rate of DNA svnthesis was determined by labeling for 1 h with 14C-TdR (0.5 ,Ci! meq 25 AiCi/mAM) at the times indicated. The first shift was done when the cells reached confluence at 32 C (0), the second was done 24 h later (M). wtSV3T3 cells were also shifted from 32 to 39 C approximately 1 day after confluence (A--- -A). tsH6-15 cells at 32 C, (O---0). wtSV3T3 at 32 C (A). A /-
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Days FIG. 4. Effect of shift-up on the cell density of the ts transformants at 39 C. tsH6-15 cells were plated at approximately 3 x 104 cells/cm2 in medium containing "4C-TdR (0.1 ACi, 10Ag/ml) and incubated at 39 C (-O-) and at 32 C (-A-). At the times indicated, the amount of label incorporated was determined. At day 5, several plates growing at 32 C were shifted to 39 C (-*-). Medium containing the same amount of TdR with the same specific activity was changed every 3 days.
VOL. 11, 1973
705
TEMPERATURE-SENSITIVE SV40-TRANSFORMED CELLS
by labeling the cells with radioactive thymidine in a continuous fashion from the time of plating, and then determining the radioactivity present in the washed cell layer after trichloroacetic acid precipitation. With such a technique, the total counts per minute per culture are essentially an indication of cell number. When multilayer cultures of tsSV3T3 cells were shifted from 32 to 39 C, the radioactivity per culture (or the cell number) increased for about 1 day, then a precipitous drop was observed. The loss of cells continued for about 3 days, after which a cell density similar to that obtained in cultures plated at 39 C was reached. This loss involved up to 80% of the cell population. wtSV40 transformants did not show this behavior. The ts cells which upon shift-up detached from the layer were not viable. In one experiment, 5 x 105 cells which had detached during a 30-h interval after shift-up were collected by centrifugation from the culture medium and were replated at 32 C. Their efficiency of plating was less then 10-4 (Table 1). To determine whether the loss of viability followed detachment, the following experiment was performed. tsH6-15 cells growing in plastic bottles at 32 C were shifted to 39 C after they had reached high densities. The bottle was filled with culture medium and inverted so that the cell layer was now on the upper surface of the culture bottle. Under these conditions, detached cells would have to travel only a short path before settling down on the empty surface and thus may not be exposed to the deleterious effect of floating in the medium. A total of
about 12 colonies per bottle appeared on the surface opposite the main cell layer while the number of cells lost was on the order of 2 x 106. Therefore, it would seem that, under these conditions, detachment of cells from the layer is accompanied by loss of viability, and death does not result solely from floating freely in the medium. The majority of the cells which did not detach remained viable. This is shown in Fig. 5, where it can be seen that when dense cultures that had been shifted to 39 C were reshifted to 32 C, cell growth resumed and the cultures attained a high density again. The experiments suggest that the ts transformants at 39 C can survive only when attached to the surface of the culture vessel. Cell detachment could be due to changes in the surface resulting in decreased adhesiveness of one cell to another. It could be asked whether normal 3T3 cells also possess this property. To approximate a situation in which normal 3T3 are grown to high densities and then shifted to a "normal" condition, we plated 3T3 cells at densities higher than their saturation and determined whether the cell number increased, remained constant, or decreased after plating. Under those conditions, no growth would be required in order to maintain the density at which the cells were plated. It was found that when 3T3 cells were plated at 5 x 106 cells/60 mm-petri dish (about five times their saturation density) the number of cells attached never reached the number of U
TAmz 1 Survival of tsSV3T3 cells detached at 39 C after shift-up of dense cultures"
/ i
Determination I
ts Cells ts Cells detached
Plating Avg Cell no. no. of efficiency (%) plated colonies
10'
1,000 500
100 Control ts cells, growing at 39 C, trypsinized
1,000 500 100
4 0 0 0
0.004 0 0 0
> 100 > 10 > 100 > 20 25 25
atsH6-15 cells were grown to high density at 32 C (i.e., 4 days past confluence), and then shifted to 39 C after medium change. Thirty hours later the medium was collected and the cell layer was washed once with medium. The cells contained in the original and the washing medium (>5 x 10' cells per 50-mm petri dish culture) were plated at 32 C.
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FIG. 5. Survival of the ts transformants after shiftup. The experiment was conducted as described under Fig. 4, except that at day 11, 6 days after shift-up, a number of replica plates were shifted again to 32 C (-U-). Medium was changed at the times of shift and 3 days thereafter.
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RENGER AND BASILICO
J. VIROL.
cells plated and in a few days was down to the characteristic saturation density of this line. This appeared to be due to the inability of some cells to attach, as well as to the loss of some attached cells from the layer (Fig. 6A). A similar result was obtained when 3T3 cells were grown to high densities with medium containing 50% serum (4, 12) and then shifted to medium containing the "normal" (10%) serum concentration (Fig. 6B). It might be concluded that under these conditions, 3T3 cells exhibit a behavior
comparable to that of the ts transformants upon shift to 39 C. The question arises then of whether the inability of tsSV3T3 to form colonies on a monolayer of 3T3 cells at 39 C (9) could be explained on the basis of lack of attachment to the cell monolayer. To test this possibility cells were labeled with 3H-thymidine, and plated on top of a confluent monolayer of 3T3 cells at 39 and 32 C, in non-radioactive medium. The radioactivity present in the cell layer was determined at various days after plating. It can be A B seen (Table 2) that all the cells tested seemed to R, attach to the 3T3 monolayer, and, furthermore, no substantial loss occurred up to 5 days after 4 I? Q plating, irrespective of whether the cells eventually formed colonies. It can thus be concluded 3that the inability of tsSV3T3 cells to form col-o onies on 3T3 monolayers at 39 C is not due ^ to lack of attachment, but is more likely due -C \1 to inhibition of cell division. The apparent U? discrepancy between these results and those _-_-reported above can probably be explained by O12 3 45 0 12 3 45 comparing the different cell numbers involved. Days After Plating Days after Serum Change In the shift-up experiments or in the plating of FIG. 6. A, Saturation density of 3T3 cells plated at 3T3 at high densities, large numbers of cells different densities. Cells (2.5 x 10-' [-A-] and 5 x (3-4 times the saturation density) are involved. 106 [-O-]) were plated in 50-mm petri dishes. The In the plating on top, 20,000 cells were plated amount of cells attached was determined at the times indicated by trypsinization and counting. The me- over a monolayer of approximately 106 cells. dium contained 10% serum and was changed at day 3. This suggests that the cell detachment is a B, Saturation density of 3T3 cells after shift from population phenomenon. medium with 50% to medium with 10% serum. Cells Origin of the ts transformants. As menin the amount of 2.5 x 105 were plated in 50-mm petri tioned before, the ts transformed lines used so dishes in medium containing 50% serum and grown to far have been isolated from stocks of 3T3 which saturation. Two days later they were washed and had been infected with mutagenized SV40 (9). incubated with medium containing 10% serum. The amount of cells attached was determined as in A at These virus preparations contained some ts the times indicated. The dashed line (---) indicates mutants since we have been able to isolate two the density reached by the cells when growing in 10% ts mutants from the screening of approximately 300 plaques isolated from the stocks. However, serum. Medium was changed at day 3. CL
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TABLE 2 Attachment of tsSV3T3 cells to 3T3 monolayersa Culture at 32 C (counts/min)
Cultures at 39 C (counts/min) Cells
3T3 wtSV3T3 tsH6 tsH6-15
No. of cells plated
Counts per minute
20,000 20,000
12,500 10,000
20,000 20,000
7,900 9,300
plated
1 day
3 days
6 days
11,400 11,100 7,800 9,600
12,000 9,900 7,800
9,300 6,500 NDC
Colony" forma-
tion -
+ -
Colony"
1 day
3 days
6 days
forma-
10,500 12,000 10,000 10,500
10,800 8,600 9,600 9,350
9,000 6,700
+
ND
+
tion -
+ 7,500 6,800 a Cells were grown for 2 days at 32 C in the presence of tritium-labeled TdR (1 MCi, 2 jig/ml), followed by a 24-h chase with 10 jig of cold TdR per ml. They were then taken off the plate with ethylenediaminetetraacetic acid and washed by centrifugation, and the specific radioactivity was determined. The cells were plated on a monolayer of 3T3 cells in 50-mm petri dishes. IColonies visible under low-power microscope 5 days after plating; (-) fewer than 100 per plate, (+) too many to count; practically confluent. c ND,
not done.
ND
VOL. 11, 1973
TEMPERATURE-SENSITIVE SV40-TRANSFORMED CELLS
the virus rescued by fusion from the ts transformants tested was invariably wild type, suggesting that we were dealing with a host cell, rather than a viral mutation. To better clarify this point, we undertook the isolation of tsSV3T3 lines from 3T3 stocks which had been infected with wt, non-mutagenized SV40. We have now been successful in isolating two ts transformed lines from those stocks. These lines are T-antigen positive at 32 and 39 C, display high saturation density at 32 C, low density at 39 C, and plate on 3T3 monolayers with much higher efficiency at the low than at the high temperature (Table 3). In essence, they are very similar to the lines isolated after infection with mutagenized virus. DISCUSSION The results presented in this paper further characterize the properties of our lines of tsSV3T3, particularly with respect to their origin and to the number of changes which occur in these cells when growing at different temperatures. The fact that we have been able to obtain similar mutants from stocks of 3T3 which have been infected with non-mutagenized wild-type SV40 further supports the idea that these cells carry a host mutation which interferes with the expression of transformation at 39 C. As mentioned before, most parameters of transformation are ts in these cells. An unexpected property of the ts transformants was found during shift-up experiments. It appears that at 39 C, they cannot maintain high densities even if shifted to this temperature TABLE 3 Characteristics of newly isolated ts
transformantsa Saturation
Cells Cls
density (Ratio 39 C/32 C)
3T3 wtSV3T3 tsH6 tsS29 tsS28
0.80 0.85 0.14 0.23 0.19
Colonyforming on ability 3T3 mono-
T-antigen'
layer" (Ratio 39 C/32 C)
39 C -
-
1 0.04 0.05 0.08
+ + + +
+ + + +
32 C
a Cells: 3T3: normal untransformed parent cell line; wtSV3T3: SV40 transformed 3T3 (SV-101); tsH6: previously described tsSV3T3 (9); tsS29, tsS28: newly isolated tsSV3T3, using non-mutagenized SV40 virus for initial infection. ' (+), more than 99% of the cells positive; (-), no cells positive.
707
after attaining such densities at 32 C. When dense multilayer cultures of tsSV3T3 cells are shifted from 32 to 39 C, DNA synthesis declines to very low levels within 24 to 36 h. At about 24 h the cultures start to shed a large number of cells into the medium. These cells are not viable and the available data suggest that their death does not follow detachment, but precedes it or at least takes place at the same time. It may be important to note that: (i) the shedding of dead cells ends at a point in which the cultures have reached the density characteristic of their saturation at 39 C, and the cells remaining are fully viable, as they can restart multiplying after the shift to 32 C; (ii) the phenomenon seems to occur after inhibition of DNA synthesis has taken place, at about the same time surface changes can be detected (5a); (iii) it seems to be a population phenomenon, at least insofar as the ability of a small number of tsSV3T3 cells to attach to a monolayer of normal cells at 39 C does not seem to be impaired. We can compare this behavior to that of normal 3T3 cells when plated at very high densities, or when shifted to normal serum concentrations after a period in medium containing 50% serum. In both cases the number of cells attached to the plate declines until it reaches the number characteristic of the saturation density of 3T3 cells in the presence of 10% serum (4, 12). These observations suggest two possible interpretations of the phenomenon we observed. One of these would ascribe it to surface changes. The surface of tsSV3T3 cells changes upon shifting from 32 to 39 C (5a), and it could be assumed that the surface configuration characteristic at 39 C is such that the cells cannot maintain contact to one another in a multilayer culture, but need a solid substrate on which to attach and spread. The sudden change from one state to another in the absence of such a substrate could cause loss of viability. This the data on the surface changes (5a) and by the fact that the cells attached to the culture vessel remain viable. A second interpretation is that the phenomenon of cell detachment upon shift-up would be the result of changes in the ability of tsSV3T3 to utilize serum factors. The saturation density of normal 3T3 cells has been shown to depend on the serum concentration in the medium (4, 12), and switching 3T3 cells from 50 to 10% serum causes cell loss from the layer. It could be thought that a drastic change in the serum requirements of tsSV3T3 during the shift from 32 to 39 C could make them unable to maintain a high saturation, as that would require very
708
RENGER AND BASILICO
high serum concentrations at 39 C. We have, in fact, been able to prevent a substantial cell loss upon shift-up by supplementing the medium with 50% serum. The serum could contribute survival factors, or factors which stimulate the ability of the cells to utilize nutrients in the medium (3). In that case, the cell concentration per plate would decrease to the point in which serum factors are sufficient to maintain the viability of the cells present. The effect would be similar to that obtained by switching 3T3 cells from medium with 50% serum to medium with 10% serum, and it would imply that the serum properties of our cells are different at 32 C and at 39 C. We previously reported (9) that we could not detect substantial differences in the serum requirements of tsSV3T3 cells at 32 and 39 C. However, more recently Holley and co-workers, using more sensitive techniques, have been able to demonstrate that the tsSV3T3 cells display some temperature sensitivity also with respect to this property, i.e., their serum requirements at 32 C are like those of transformed cells, while at 39 C they resemble more those of normal 3T3 cells (R. W. Holley, personal communication). At present, both interpretations seem possible, since the close correlation between surface and growth properties of these cells make it difficult to separate the two types of phenomena. It is evident, however, that all of these changes follow a primary change in these cells which occurs when they are shifted from one temperature to another. The experiments we described suggest that the low saturation density of normal 3T3 cells in culture (or of our ts transformants at 39 C) is maintained phenotypically by at least two blocks. Inhibition of DNA synthesis generally prevents cell division after formation of a monolayer, but, if this control fails, high densities cannot be reached since the cells cannot attain or maintain a multilayer state. Whether both these properties can be ascribed to the same major determinant (surface or ability to utilize serum factors) is not yet clear, but it is clear that they are distinct phenomena. Thus, cells could lose contact inhibition of DNA synthesis, without being able to reach high densities (10). To summarize, we have discovered an unexpected property of tsSV3T3 cells, that of being temperature sensitive even for maintenance of high cell densities, and not only for the capacity of attaining them. The available data suggest that this property could be due to changes in the
J. VIROL.
surface or in serum requirements. The mechanism of cell death which accompanies this phenomen is still obscure. However, the present data show quite conclusively that the host mutation our tsSV3T3 cells carry affects a number of parameters of malignant transformation, and possibly all of them. ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant CA-11893 from the National Cancer Institute, and by grant VC-99A from the American Cancer Society. H.C.R. was a fellow of the Jane Coffin Childs Memorial Fund. C.B. is a scholar of the Leukemia Society.
LITERATURE CITED 1. Basilico, C., Y. Matsuya, and H. Green. 1970. The interaction of polyoma virus with mouse-hamster somatic hybrid cells. Virology 41:295-305. 2. Eckhart, D., R. Dulbecco, and M. M. Burger. 1971. Temperature dependent surface changes in cells infected or transformed by a thermosensitive mutant of polyoma virus. Proc. Nat. Acad. Sci. U.S.A. 68:283-286. 3. Holley, R. W. 1972. A unifying hypothesis concerning the nature of- malignant growth. Proc. Nat. Acad. Sci. U.S.A. 69:2840-2841. 4. Holley, R. W., and J. A. Kiernan. 1968. "Contact inhibition" of cell division in 3T3 cells. Proc. Nat. Acad. Sci. U.S.A. 60:300-304. 5. Martin, G. S. 1970. Rous sarcoma virus: a function required for'the maintenance of the transformed state. Nature (London) 227:1021-1023. 5a. Noonan, K. D., H. C. Renger, C. Basilico, and M. M. Burger. 1973. Surface changes in temperature-sensitive simian virus 40-transformed cells. Proc. Nat. Acad. Sci. U.S.A. 70:347-349. 6. Otten, G., J. Bader, G. S. Johnson, and I. Pastan. 1972. A mutation in a Rous sarcoma virus gene that controls adenosine 3,5-monophosphate levels and transformation. J. Biol. Chem. 247:1632-1633. 7. Pollack, R. E., H. Green, and G. J. Todaro. 1968. Growth control in cultured cells: selection of sublines with increased sensitivity to contact inhibition and decreased tumor-producing ability. Proc. Nat. Acad. Sci. U.S.A. 60:126-133. 8. Renger, H. C. 1972. Retransformation of tsSV40 transformants by murine sarcoma virus at non-permissive temperature. Nature N. Biol. 240:19-21. 9. Renger, H. C., and C. Basilico. 1972. Mutation causing temperature-sensitive expression of cell transformation by a tumor virus. Proc. Nat. Acad. Sci. U.S.A. 69:109-114. 10. Scher, C. D., and W. A. Nelson-Rees. 1971. Direct isolation and characterization of "flat" SV40-transformed cells. Nature N. Biol. 233:263-265. 11. Todaro, G. J., and H. Green. 1963. Quantitative studies on the growth of mouse embryo cells in culture and their development into established lines. J. Cell Biol. 17:299-313. 12. Todaro, G., Y. Matsuya, S. Bloom, A. Robbins, and H. Green. 1967. Stimulation of RNA synthesis and cell division in resting cells by a factor present in serum. p. 87-101. Wistar Symp. Monogr.
