DNA content distribution of mouse cells ... - Wiley Online Library

Cytometry 16:138-143 (1994)

0 1994 Wiley-Liss, Inc.

DNA Content Distribution of Mouse Cells Following Infection with Polyoma Virus' John M. Lehman, Judith Laffin, and Thomas D. Friedrich Department of Microbiology, Immunology and Molecular Genetics, Albany Medical College, Albany, New York 12208 Received for publication August 11, 1993; accepted October 22, 1993

Infection of primary to tertiary mouse embryo fibroblasts or mouse kidney cells with polyoma virus leads to stimulation of cellular DNA synthesis. When either confluent or growing mouse cells were infected, the monolayer cells were found to accumulate cells with a DNA content of S and G,/M phases of the cell cycle as assayed by flow cytometry. A similar pattern of DNA content was also observed in cells in the supernatant, which are probably cells replicating virus and dying. When compared with control cells, the infected monolayer and supernatant cells exhibited a population (5-27%) with a

The papovaviruses induce permissive and nonpermissive cells into DNA synthesis (19). Early studies with mouse embryo cells following infection with polyoma (Py) demonstrated a n increased incorporation of 3H thymidine into DNA of which two thirds was in cellular DNA vs. viral DNA (4).Further, early studies suggested that the induction of cellular DNA synthesis required expression of the viral DNA (19). Recent studies have suggested that virus adsorption may suffice to induce c-fos, c-jun, and c-myc, which is usually followed by initiation of cellular DNA synthesis. Therefore, the binding of virus to the cell membrane may set in motion events that lead to the stimulation of cellular DNA synthesis (8,ZO). At a later time point, a second stimulation occurs which is probably dependent upon the polyoma T antigens (8). Studies with mutant Py virus have suggested that the second phase of c-rnyc and c-fos accumulation may be regulated by the small T either alone or in combination with the large T antigen (20). When Ogris et al. (17) transfected plasmids that contained the large or small polyoma T antigens under the control of a dexamethasone inducible mammary tumor virus promoter and a selective marker into 3T3 cells, the isolated cells were induced to express the large or small T antigen. The results demonstrate that neither large or small T antigen alone stimulated DNA

>G, DNA content. The increase in DNA content of these >G, cells was calculated to be an average of 26.7%,which is probably due to viral DNA. Polyoma contrasts with another papovavirus, SV40, which stimulates cells into DNA synthesis, with the majority of cells attaining a >G,/tetraploid DNA content, suggesting that there are differences in polyploidization between these two viruses. 0 1994 Wiley-Liss, Inc.

Key terms: Polyomavirus, cellular DNA synthesis, flow cytometry, DNA content

synthesis; however, if both T antigens were expressed in the same serum starved 3T3 cell, DNA synthesis was induced. These studies are in contrast with another papovavirus SV40, which stimulates cellular DNA synthesis following expression of the large T antigen (11,14-16). Studies with viral mutants and viral DNA suggest that functions of the large T antigen are responsible for the induction of cellular DNA synthesis. When DNA content analyses were performed with flow cytometry, both permissive and nonpermissive cells are induced into multiple rounds of DNA synthesis without mitosis which produce a cell with a >G, DNA content (tetraploid S and tetraploid G,/M) (11,14-16). Further, a temperature-sensitive mutant of SV40, tsA 30, was unable to initiate a second round of DNA synthesis a t the nonpermissive temperature (405°C) identifying a T antigen function necessary to bypass G,/M controls and initiate a second round of DNA synthesis (6).

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'This work was supported by grant CA-41608 from the National Cancer Institute. 'Address reprint requests to John M. Lehman, Dept. of Microbiology, Immunology & Molecular Genetics, A-68, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208.

DNA DISTRIBUTION AFTER POLYOMA INFECTION

The question arises as to whether the stimulation of cellular DNA by Py virus is induced into single or multiple S phases without mitosis, leading to a population of >G, or tetraploid cells in a similar manner to SV40. These studies analyzed the DNA content pattern of mouse embryo fibroblasts and mouse kidney cells infected with Py virus. The DNA synthesis was assayed with flow cytometry (FCM), since this technique allows a n analysis of the distribution of cells about the cell cycle, GI, S, G,/M, and >G,. Numerous experiments demonstrated a DNA content distribution which suggested stimulation of DNA synthesis into the S, G,, and M phases of the cell cycle. However, no evidence was obtained to suggest that cells were stimulated into multiple S phases without mitosis, leading to a >G, population.

MATERIALS AND METHODS Mouse embryo fibroblasts (MEF) were prepared from 15 to 17-day-old embryos of Swiss-Webster mice as previously described (1). Mouse kidney (MK) cells were prepared from 1-day-old mice. The cells were subcultured with trypsin-versene and grown in MEM (Gibco, Grand Island, NY) supplemented with 5% fetal bovine serum (FBS; Gibco, Grand Island, NY) and 100 pgiml of penicillin and 100 units of streptomycin (15,16) at 37°C in 5% CO, incubator. Wild type Py virus was prepared and titered as previously described (1,2). The cells were infected with 50 plaque forming units (PFU)/ cell for a 1 h r adsorption. Following adsorption the virus was removed and MEM with 5% FBS was added. The control cultures were refed with MEM 5% FBS, and both infected and control cultures were harvested at the times indicated. To determine the number of cells expressing T antigen, the cells were grown on coverslips, fixed with 70% acetone and 30% methanol at -20"C, and stained by indirect immunofluorescence with a polyclonal antibody described by Silver et al. (18). The CV-1 cells, SV40 virus, and the infection procedure have been described previously (6,7,15). DNA content analysis was performed by FCM analysis on single cells that were trypsinized and fixed in methanol a s previously described (10). In three of eight experiments, supernatant cells were harvested by centrifugation, resuspended in phosphate buffered saline, and fixed in methanol. The cells were treated with RNase (Sigma) and with propidium iodide (PI) as previously described (10). Stained cells were analyzed with a n Ortho cytofluorograph I1 model 50 H-H and 2151 data analysis system using a n Omnichrome air cooled argon laser (model 532) a t a wave length of 488 nm and 20 mW of power. The instrument was aligned immediately before use with fluorescent microspheres (Polysciences 9847, Warrington, PA) and PI stained mouse lymphocytes. For analysis no filter was used for light scatter, but a barrier filter (640 nm long pass) was used for red. Data were collected and stored in a two parameter cytogram. First a gated population of light scatter vs. red fluorescence (DNA) eliminated noncel-

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lular material. Cells passing through this gate were analyzed using red area vs. red peak for selection of single cells. For data analysis of the DNA content distribution, we gated the control population for GI, S, G,/M, and >G, cells to determine the number and percentage in each phase of the cell cycle. The gates were set on the GI peak, the ascending and descending curves. The S phase population was measured from the descending GI peak to the ascending curve of the G, phase. The Gz phase was a peak and a n arbitrary ascending curve was drawn for his peak. All cells above the Gz phase were considered the >G, population. The corresponding (time point) infected population was similarly gated and the number and percentages determined. This analysis allowed a comparison of percentage of cells in the various stages of the cell cycle a t various times post-infection. Ten thousand cells were analyzed by FCM for each time point.

RESULTS These studies were performed on primary to tertiary mouse embryo fibroblasts and mouse kidney cultures. Early passage cells were used since later passages become refractory to infection with Py virus. Six experiments were performed with MEF and in two of these experiments the supernatant cells were harvested. Two additional experiments were performed with the MK cells, and in one experiment the supernatant cells were harvested. The cells were infected and followed a t various times to determine the DNA content changes as the cells progressed through the infection cycle. With multiplicities of 50 PFUkell, approximately 3040% of the cells were expressing T antigen a t 48 h post-infection, and this increased to >80% at later time points. To determine the influence of growth state following viral infection, growing and confluent uninfected cultures were examined by FCM. There was a significant number of cells in the S phase at early times in the growing cultures; however, this level decreased with time (Table 1, see S phase). By 96 h after mock infection, growing and growth arrested control cultures had 75-79% of their cells in GI and 2-3% in >G,. This distribution continued for the remaining time points. Therefore, the control population of mouse cells had a normal cell cycle DNA content distribution. To enhance the infection efficiency, growing, subconfluent cultures were infected and examined for cell cycle distribution (Table 1). At the early time points (24, 48, and 72 h) the values obtained are representative of replicating cells in both control and infected cultures. When the later time points were assayed (120 and 144 h) the infected population had accumulated greater than 50%of the cells in the S and G,/M phase of the cell cycle. This is evident when the histograms of the DNA content were compared between the control and infected populations (Fig. 1). The Py infected cells underwent DNA synthesis with the accumulation of cells in the S and G,/M phases of the cell cycle. Growth arrested cultures were infected with Py and

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LEHMAN ET AI,.

Table 1 Percentage of Mouse Embryo Fibroblasts Distributed Through the Cell Cycle at Various Times Post-Infection"

Table 2 Percentage of Cells Distributed Through the Cell Cycle at Various Times Post-Infection"

Phase of the Cell Cycle 24 h 48 h 72 h 96 h 120 h 144 h

C I C I C I C I C I C I

GI

S

45 52 70 61 66 58 74 36 76 40 75 38

22 15 16 17 11 16 7 23 8 20 9 23

G2 26 26 11 17 19 19 17 31 14 30 14 27

G,

>G2 7 7 3 5 4 7 2 10 2 10 2 12

"Growing, subconfluent MEF were infected with 50 PFUi cell and harvested by trypsinization at various times postinfection. These data were derived by placing gates around the populations and calculating the percentage of the total (see Fig. 1).Ten thousand cells at each time point were assayed. C = control cells; I = infected cells, monolayer.

96 h

C I

125 h

C I

164 h

C I

S S

S

79 36 7 76 32 14 78 26 16

Phase of the S 3 21 30 3 25 28 3 24 24

>G, 3 16 24 3 15 21 2 18 25

"Confluent mouse kidney cells were infected with 50 PFUi cell of polyomavirus. The cells were growth arrested 4 days prior to infection. At the time points indicated, cells were trypsinized, fixed, and stained as described in Materials and Methods. The floating cells were harvested, pelleted, fixed, and stained for FCM as described in Materials and Methods. C = control; I = infected, monolayer; S = supernatant (floating). 120HR

the attached cells assayed for cell cycle distribution. At 96, 125, and 164 h post-infection, 26-36% of the population was in G, with 15-18% in >G,. S phase contained 21-25%. Thus cells were observed in the S and then G,/M phase of the cell cycle during the infection (Table 2). The G, population decreased and did not increase a t later time points, suggesting that the majority of G, cells did not progress through mitosis into the G, phase of the cell cycle (Table 2). However, some cells may have undergone mitosis and a subsequent S phase, retaining a diploid DNA content. Both the confluent and growing populations demonstrate the stimulation into DNA synthesis by the Py virus. The infected cells move into the S and G,/M phase of the cell cycle, with a small number of cells acquiring a n increase in the G2 amount of DNA. At this point, the cells are making viral DNA, late protein, and virions. These cells are dying, detaching from the monolayer, and floating in the supernatant (5). In the Py infected culture, cytopathic effect (CPE) progressed, the cells detached from the monolayer, and clumped a s they floated in medium (5). A comparison of the DNA content of the floating (supernatant) cells to monolayer cells is shown in Figure 2a and 2b, and Table 2. The majority of the floating cells were in S, G2/M, and >G,, with some small fragmented material; however, this pattern of accumulated S and G,/M cells was similar in the two samples. The cells with the >G2 amounts of DNA were shifted approximately 10-12 channels above the median for the G2 peak of DNA content (Fig. 2b). If a gate is set around the cells with >G, amount of DNA, this percentage can be calculated (Table 3, Fig. 2c). This provides the number of cells contained in the >G, population. Further, these >G, cells have a DNA content that is increased and can be

cell cycle G, 15 28 38 18 28 33 17 32 35

1441iR

CONTROL

INFECTCD

DNA CONTEIdI

FIG.1. DNA distribution of Py-infected and control MEF a t 96,120, and 144 h. The cells were harvested from the monolayer, fixed, and stained with PI as described in Materials and Methods. The x-axis is DNA content with a linear increase. The y axis is the number of cells analyzed. To determine the percentage of cells in each stage of the cell cycle, a gate was chosen to determine the number of cells in GI, S, G,/M, and >G2 for the control cells; then the gates were used to determine the number of infected cells in these populations. This allows a comparison of cells in each stage of the cell cycle between infected and control populations. Ten thousand cells were analyzed for DNA content at each time point by FCM.

determined by comparing the shift in channel number (x axis, DNA content) by placing gates around the G2 and >G, populations. From these data the mean can be calculated for these populations. The formula [(mean >G2 - mean G2)/ mean G,] x 100 will provide the percentage increase in DNA for the >G2 cells. This will provide the amount of increase in DNA (% increase) which theoretically would be the increase due to the additional viral and/or


Table 3 Mean of the G2Phase and >Gz Phase Infected Population and Percentage Increase of >Gz Cells" Experiment Experiment 1

Experiment 2

Hrs. P.I. 72 96 120 144 72 96 120 72

Mean G, 35 35 35 35 30 30 30 29

Mean

%

>G, 42.1 42.2 43.1 42.6 40.3 39.6 39.4 37.8

Increase 20.5 20.6 23.1 21.7 34 32 31

Experiment 3 30 aGates were set around the G, phase population of polyoma infected mouse embryo fibroblasts to determine the mean channel number (DNA content) for each population. To calculate the percentage increase in the DNA for the >G, phase population, the following formula was utilized: [(mean >G,mean G,)/mean G,] x 100. P.I. = post-infection.

cellular DNA (average of 26.7%; Table 3). The increase in DNA of these cells amounted to 20-30%, which compared favorably with the early studies of Dulbecco et al. (41, who demonstrated that approximately two thirds of the newly replicated DNA is cellular and that the newly replicated viral DNA accounts for approximately 30% of the newly synthesized total DNA.FCM, while unable to quantitate the cellular vs. the viral DNA,is able to quantitate the total amount of DNA per cell. These studies demonstrate the movement of cells into the S and G2 phases of the cell cycle, where these cells accumulate and replicate virus. For comparison a CV-1-SV40 infected population at 0,24, and 48 h post-infection is shown in Figure 2d. These results demonstrate the increase in the >Gz (tetraploid) population as the cells progress through the infection cycle. At 24 and 48 h post-infection, the >G2 population is 12% and 68%, respectively, with the majority of the cells expressing T antigen, 84% a t 24 h and 92% a t 48 h.

DISCUSSION The papovaviruses are unique in their ability to stimulate permissive and nonpermissive cells into DNA synthesis (19). This property may be a n important step in the neoplastic conversion of cells with these viruses. In the studies presented, polyoma stimulated both growing and contact-inhibited cells into DNA synthesis a s assayed by FCM. The majority of the cells remain with S and G2/MDNA content (Figs. 1and 2). Since the number of GI cells decreases, the majority of cells are either blocked in S and Gz, or are lost into the supernatant. With this analysis, we cannot rule out that a few percentage of cells undergo mitosis into GI, however, these cells must cycle into S and G2,and become blocked in these stages. Further, the DNA content distribution did not demonstrate a large number of cells with >G, (tetraploid-polyploid) DNA content, as

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is observed with SV40 infection of permissive cells (15) (see Fig. Id). Polyoma CPE in mouse embryo fibroblasts is observed as patches of small rounded cells which detach into the supernatant or stay attached to the monolayer by small filaments. These supernatant cells may be collected by washing the monolayer and pelleting these cells by centrifugation throughout the experiment. We are confident that these analyses have collected all the cells both attached and supernatant; therefore, these results have included all infected cells in the analysis. The DNA patterns of the supernatant cells were similar in DNA content to the monolayer cells, except that the majority were in S and G,/M. A small number of cells have a DNA content of >G,. In fact, this increase in DNA is observed as a shift to the right of the descending Gz/M peak and would be the total of cellular DNA and viral DNA synthesized during infection. For both the Py and SV40 lytic infection, the actual percentage increase in viral and cellular DNA replicated needs to be determined. We are attempting this analysis using the separation of cellular and viral DNA by the Hirt extraction procedure to determine this increase in viral DNA.The cellular DNA will be analyzed by using the FISH procedure for specific cellular DNA to determine gene amplification following infection. These FCM studies demonstrate the DNA content changes with the movement of cells into the S and Gz/M phase of the cell cycle. The number of G, cells decreases with time; however, some cells may undergo mitosis into the G, phase of the cell cycle. In previous studies, cells obtained from tumors induced by Py in the Syrian hamster, and Syrian hamster transformed cells in vitro, exhibited a diploid or pseudodiploid karyotype (1,2). There was a n increase in the number of tetraploid/polyploid cells; however, this was only 3-15%. Therefore, the majority of neoplastic cells induced by polyoma were near-diploid. This is in contrast with SV40 induced tumors and transformed cells, which exhibit a near-tetraploid chromosome number distribution (12,13). Previous studies have shown that mouse embryo fibroblasts and mouse macrophages are induced into multiple rounds of cellular DNA synthesis following infection with SV40 (12,16). Therefore this suggests that the lack of >G2 cells following infection with Py is due to the virus and not the cell type. It has been shown definitively that the SV40 T antigen is responsible for the stimulation of cell DNA synthesis following infection. More recent studies have suggested that multiple regions of the T antigen may be involved, which include the p53 binding site, the pRB binding site, and a n activity in the DNA binding region (3). Further, the hypophosphorylation of pRB may be involved in the generation of the tetraploid cells following infection with SV40 (6). However, recent evidence suggests that polyoma infected mouse cells do not accumulate hypophosphorylated pRB (9). The study of these two viruses may provide a n understanding of the mechanism for the regulatory

LEHMAN ET AL.

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a I6411R

9GllR

1

C O N T R 01.

w Ld m I

z 3 INF E C iE U

J J w

V

SUPERNAIE

DNA CONlENT

b

1

SO

100

DNA CONTENT

C

DNA CONTENT

FIG.2. DNA distribution of Py-infected growth arrested mouse kidney (MK) cells and SV40 infected CV-1 cells. a: Polyoma infected and uninfected mouse kidney cells were harvested at 96, 125, and 164 h. The cells were harvested from the monolayer and supernatant, and processed was for flow cytometry as described in Materials and Methods. Control MK cells were harvested from monolayer. The data were analyzed as described in Materials and Methods. b The DNA distribution of attached (Mono) and supernatant (Sup) cells a t 144 h postinfection. The supernatant cells were harvested, pelleted, resus-

DNA CONTENT

pended, fixed, and stained with PI. The monolayer cells were trypsinized, fixed, and stained with PI. c: DNA distribution of uninfected and infected (shaded) cells at 144 h post-infection. Note the increased number of cells in S, G,, and >G2 phase in the infected population compared to the control population. d SV40 infected CV-1 cells a t 0, 24, and 48 h post-infection. At 24 and 48 h post-infection, 84% and 96%of the cells were expressing T antigen. The percentage of cells in >G2 was 3% at 0 h, 12%a t 24 h, and 68% at 48 h post-infection.


controls of cellular DNA synthesis and how a viral proteinb) may stimulate and modify this cellular regulatory process. A considerable amount of interest has focused on the phenomenon of viral stimulated cellular DNA synthesis, since an understanding may suggest a pathwayk) to a neoplastic cell.

ACKNOWLEDGMENTS The authors would like to thank Ms. Lynn Ashline and Ms. Emilee Dickerson for technical help, and Ms. J o Ann D’Annibale for typing the manuscript.

LITERATURE CITED 1. Defendi V, Lehman JM: Transformation of hamster embryo cells in vitro by polyoma virus: Morphological, karyological, immunological and transplantation characteristics. J Cell Comp Physiol 66:315-410, 1965. 2. Defendi V, Lehman JM: Biological characteristics of primary tumors induced by polyoma virus in hamsters. Int J Cancer 1:525540,1966. 3. Dobbelstein M, Arthur AK, Dehde S, vanZee K, Dickmanns A, Fanning E: Intracistronic complementation reveals a new function of SV40 T antigen that cooperates with Rb and p53 binding to stimulate DNA synthesis in quiescent cells. Oncogene 7:837849,1992. 4. Dulbecco R, Hartwell LH, Vogt M: Induction of cellular DNA synthesis by polyoma virus. Proc Natl Acad Sci USA 53:403-410, 1965. 5. Eddy BE: Polyoma virus. In: Virology Monographs, Vol. 7, Gard S, Hallaner C, Meyer KF (eds).Springer-Verlag,New York, 1969, pp 1-114. 6. Friedrich T, Laffin J, Lehman JM: Simian virus 40 large T antigen function is required for induction of tetraploid DNA content during lytic infection. J Virol 66:4576-4579, 1992. 7. Friedrich T, Laffn J, Lehman JM: Hypophosphorylated retinoblastoma gene product accumulation in SV40 infected CV-1 cells acquiring a tetraploid DNA content. Oncogene 8:1673-1677, 1993.

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8. Glenn GM, Eckhart W: Transcriptional regulation of early-response genes during polyomavirus infection. J Virol 64:21932201,1990. 9. Khandjian EW, Tremblay S: Phosphorylation of the retroblastoma protein is modulated in mouse kidney cells infected with polyomavirus. Oncogene 7:909-917, 1992. 10. Laffn J , Lehman JM: Detection of intracellular virus and viral products. In: Methods in Cell Biology, Vol. 33, Darzynkiewicz Z, Crissman H (eds). Academic Press, New York, 1990, pp 271-284. 11. Laffin J , Fogleman D, Lehman JM: Correlation of DNA content, p53, T antigen and V antigen in Simian virus 40 infected human diploid cells. Cytometry 10:205-213, 1989. 12. Lehman J : Early chromosome changes in diploid Chinese hamster cells after infection with Simian virus 40. Int J Cancer 13: 164-172, 1974. 13. Lehman JM, Bloustein P Chromosome analysis and agglutination by concanavalin A of primary Simian virus 40 induced tumors. Int J Cancer 14:771-778, 1974. 14. Lehman JM, Defendi V: Changes in deoxyribonucleic acid synthesis regulation in Chinese hamster cells infected with Simian virus 40. J Virol 6:738-749, 1970. 15. Lehman J , Friedrich T, Lafin J : Quantitation of Simian virus 40 T antigen correlated with the cell cycle of permissive and nonpermissive cells. Cytometry 14401-410, 1993. 16. Lehman JM, Mauel J, Defendi V Regulation of DNA synthesis in macrophages infected with Simian virus 40. Exp Cell Res 67:230233, 1971. 17. Ogris E, Mudrak I, Wintersberger E: Polyomavirus large and small T antigens cooperate in induction of the S phase in serumstarved 3T3 mouse fibroblasts. J Virol 66:53-61, 1992. 18. Silver J , Schaffausen B, Benjamin T: Tumor antigens induced by nontransforming mutants of polyoma virus. Cell 15:485-496, 1978. 19. Tooze J (ed): Molecular Biology of Tumor Virus, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1981. 20. Zullo J , Stiles CD, Garcea RL: Regulation of c-myc and c-fos mRNA levels by polyomavirus: Distinct roles for the capsid protein VP, and the viral early protein. Proc Natl Acad Sci USA 84:1210-1214, 1987.