Inducers of DNA Synthesis Present During Mitosis of Mammalian Cells Lacking G1 and G2 Phases (Cell Cycle/Cell Fusion/Prematurely Condensed Chromosomes) POTU N

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Proc. Natl. Acad. Sci. USA Vol. 75, No. 10, pp. 5043-5047, October 1978 Cell Biology Inducers of DNA synthesis present during mitosis of mammalian cells lacking G1 and G2 phases (cell cycle/cell fusion/prematurely condensed chromosomes) POTU N. RAO, BARBARA A. WILSON, AND PRASAD S. SUNKARA Department of Developmental Therapeutics, The University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston, Texas 77030 Communicated by David M. Prescott, July 27, 1978

ABSTRACT The cell cycle analysis of Chinese hamster lung MATERIALS AND METHODS fibroblast V79-8 line by the premature chromosome condensation method has confirmed the absence of measurable GI and Cells and Cell Synchrony. The Chinese hamster cell line G2 periods. Sendai virus-mediated fusion of mitotic V79-8 cells (V79-8), which lacks both the GI and G2 phases in its cell cycle, with GI phase HeLa cells resulted in the induction of both DNA was kindly supplied by R. Michael Liskay, University of Col- synthesis and premature chromosome condensation in the latter, orado, Boulder, CO. V79-8 cells were grown as monolayers on indicating the presence of the inducers of DNA synthesis above Falcon plastic culture dishes in McCoy's 5A modified medium the critical level not only throughout S phase, as it is in HeLa, supplemented with 15% heat-inactivated fetal calf serum but also during mitosis of V79-8 cells. No initiation of DNA (GIBCO) in a humidified CO2 (5%) incubator at 37°. Under synthesis was observed whe-n GI phase HeLa cells were fused these conditions, this cell line had a generation time of about with mitotic CHO cells. These results indicate that the presence 10 hr (6). V79-8 cells were synchronized in mitosis by selective or absence of a GI period in the cell cycle depends on the levels of the inducers of DNA synthesis present in the cell during mi- detachment after a brief (2-hr) exposure to Colcemid (0.05 tosis. gg/ml). HeLa cells, grown in suspension culture, were routinely maintained in exponential growth by daily diluting with fresh Studies involving nuclear transplantation (1, 2) and cell fusion Eagle's minimal essential medium supplemented with heat- between cells in various phases of the cell cycle (3, 4) have inactivated fetal calf serum (10%, vol/vol), nonessential amino shown that the cytoplasm of the cells undergoing DNA repli- acids, penicillin/streptomycin mixture, sodium pyruvate, and cation contains certain factors that can induce DNA synthesis glutamine (7). For cell fusion studies, HeLa cells in Gl phase prematurely in GI nuclei when they are brought in contact with were obtained by synchronizing cells in mitosis by the nitrous each other. Preparations for the initiation of DNA synthesis in oxide block method (8) and then allowing them to divide over mammalian cells, including the synthesis of these inducers, are a period of 3 hr after the reversal of the mitotic block. A 3S-min presumed to take place during the GI phase, which immedi- pulse labeling of the Gl population with [3H]thymidine indi- ately precedes the period of DNA synthesis. However, until cated a labeling index of zero. The mitotic index was less than recently it was not clear whether the inducers of DNA synthesis 5%. reach a critical level abruptly at the Gl/S transition or accu- Cell Fusion and Cell Cycle Analysis. The cell cycle analysis mulate gradually during the GC period. On the basis of cell of V79-8 cells was performed by the use of the premature fusion studies involving HeLa cells synchronized at various chromosome condensation method (9). Mitotic and random in the populations of V79-8 cells were fused by the use of UV-inacti- points GC period, Rao et al. (5) have proposed a model vated Sendai virus to induce premature chromosome conden- regarding the availability of the inducers of DNA synthesis sation. The detailed procedures for cell cycle analysis by cell during the HeLa cell cycle. According to this model, the in- fusion have been described (9). This method of cell cycle ducers of DNA synthesis accumulate gradually during the GC analysis is based on the fact that the morphology of the pre- period, reaching a critical level by the end of this period, when maturely condensed chromosomes (PCC) of an interphase cell DNA synthesis is initiated. As DNA synthesis is completed, the indicates its position in the cell cycle at the time of fusion. level of these inducers decreases below the critical level. Kinetics of Induction of DNA Synthesis in GI Phase HeLa Therefore it is of interest to see how such a model would fit a Cells. Mitotic V79-8 cells, prelabeled with [3H]thymidine, were cell line (Chinese hamster V79-8) that has neither GC or G2 fused with HeLa cells in GI phase. Colcemid (0.5 ,g/ml) was phase in its cell cycle (6). In the Chinese hamster V79-8 cells, added to the fusion mixture along with the virus and kept in the successive periods of DNA synthesis are interrupted only by a medium throughout the experiment in order to prevent the short period of mitosis. Hence, the objective of the present study V79-8 cells from completing mitosis. The new mitotic inhibitor was to determine whether the inducers of DNA synthesis Maytansine (10) appeared to be more effective than Colcemid remain above the critical concentration throughout the cell in holding the V79-8 cells in mitosis for prolonged periods. cycle or decrease during mitosis. The results of these experi- Hence, Colcemid was replaced by Maytansine in later experi- ments indicate that the factors for the initiation of both DNA ments. synthesis and mitosis can be present simultaneously in mitotic After fusion, a small sample of the cells was deposited directly cells. on a clean slide by the use of a cytocentrifuge, fixed in a 3:1 (vol/vol) absolute methanol/glacial acetic acid mixture, and The publication costs of this article were defrayed in part by page processed forautoradiography to determine the extent of fusion. charge payment. This article must therefore be hereby marked "ad- The remaining cells were diluted with fresh medium containing vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: PCC, prematurely condensed chromosomes. 5043 Downloaded by guest on September 30, 2021 5044 Cell Biology: Rao et al. Proc. Natl. Acad. Sci. USA 75 (1978) [3H]thymidine (1.0 ,gCi/ml, 6.7 Ci/mmol) and Colcemid (0.5 ,gg/ml) and then plated in a number of 35-mm Falcon plastic .."W w*(v culture dishes. At hourly intervals, one of the dishes was - I trypsinized and the cell samples were deposited on slides, fixed, .I J and processed for autoradiography as described above. The cells 4 were stained with Giemsa and scored for the presence of label '%: SI on either the nuclei or PCC of HeLa cells residing along with 111% the mitotic chromosomes of V79-8 cells in the same cytoplasm. Chinese hamster ovary (CHO) cells, which have a GL period

of 2 to 2.5 hr, were used as a control. Mitotic CHO cells, col- -AL..' lected by selective detachment after a 2-hr exposure to Col- 9 cemid (0.5 ,gg/ml), were incubated for another 3 hr in the same 1. medium containing Colcemid and then fused with GC phase HeLa cells. After fusion, cells were incubated with [3H]thymidine and samples were taken at hourly intervals, processed, and scored as described above. About 200 cells were scored for each sample point. Cell Cycle Progression of V79-8 Cells after the Reversal of a Mitotic Block. To determine how rapidly the cells enter A S phase after cell division, we synchronized V79-8 cells in mitosis by selective detachment after a 2-hr Colcemid (0.05 ,ug/ml) block. The Colcemid was removed by washing and plating the mitotic cells in regular medium in a number of dishes. At 30-min intervals, one of the dishes was trypsinized. A small fraction of the cells was deposited on a slide by the use of cytocentrifuge, fixed, stained with aceto-orcein, and scored for mitotic index. The remaining cells in the sample were fused with mitotic V79-8 cells for cell cycle analysis by the PCC method. RESULTS Cell Cycle Analysis. The cell cycle analysis of V79-8 cells in exponential growth. by the PCC method revealed that 95.5% of the cells were in S phase, as indicated by the "pulverized" appearance of the PCC. The cells in GI phase constituted less than 1%. About 4% of the cells that exhibited PCC with G2-like morphology have completed DNA replication except for one GI , or two short regions and hence by definition should still be considered as in S phase (see Fig. 1). These data suggest that the V79-8 cells have practically no G1 and G2 in their cell cycle. Rapidity of Mitotic to S Phase Transition in V79-8 Cells. FIG. 1. PCC of V79-8 cells in exponential growth. (X1600.) (A) After the reversal of the Colcemid (0.05 ,ug/ml) block, the Product of a fusion between mitotic and S phase V79-8 cells. The PCC synchronized mitotic cells completed cell division very rapidly; are of early S phase morphology, exhibiting "pulverized" appearance. i.e., within 30 min the mitotic index decreased from 98% to 48% The darkly stained chromosomes are of the mitotic cell. (B) Product of fusion between a mitotic and two interphase cells. M = mitotic and by 60 min it was below 5%. The cycle'analysis of this pop- chromosomes, well condensed and darkly stained; G1 = G1 PCC with ulation by the PCC method at 30 min after the reversal of the one chromatid each; G2 = PCC of G2 morphology, consisting of two Colcemid block indicated that 90% of the interphase cells ex- chromatids except for one or two short segments where DNA repli- hibited PCC with early S phase morphology (Fig. 1A), while cation is still in progress as indicated by a gap (arrow). the remaining were of theG1 type. These data suggest that the transition from mitosis to S phase occurs very rapidly in V79-8 cells. chromosome condensation (Fig. 2 A and B). These results Induction of Both DNA Synthesis and Premature Chro- clearly demonstrate the presence of the inducers of both DNA mosome Condensation in GI Phase HeLa Cells by Fusion synthesis and premature chromosome condensation in the with Mitotic V79-8 Cells. Because successive rounds of DNA mitotic cells of V79-8 line. In contrast, no DNA synthesis was synthesis in V79-8 cells are interrupted by only a brief period initiated in HeLa nuclei residing in the binucleate cells formed of cell division, we decided to test whether the inducers of DNA by the fusion of mitotic CHO and HeLa GC. synthesis are present during mitosis. The fusion of mitotic V79-8 cells with HeLa cells inGI period resulted in both the induction DISCUSSION of DNA synthesis and premature chromosome condensation A reevaluation of the cell cycle parameters of the V79-8 cell line in the HeLa nuclei (Fig. 2). During the first hour of fusion, the by the PCC method has essentially confirmed the report of induction of DNA synthesis in HeLa nuclei was much more Liskay (6), who observed that this cell line has no measurable rapid than PCC induction (Fig. 3). Subsequently, both these periods ofGC and G2. parameters increased linearly as a function of time. The labeling A comparison of this cell line (V79-8) that lacksGC and G2 index in the mononucleate (U) and binucleate (UU) HeLa cells phases with another, for example, HeLa, that has four distinct remained essentially zero (U = unlabeled nucleus). In some of phases (i.e., G1, S, G2, and M) in its cell cycle may help us to the fused cells, DNA synthesis was induced but not premature understand the basis for cell cycle regulation. The parameters Downloaded by guest on September 30, 2021 Cell Biology: Rao et al. Proc. Natl. Acad. Sci. USA 75 (1978) 5045

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C D FIG. 2. Heterophasic cells resulting from the fusion between mitotic V79-8 cells prelabeled with [3H]thymidine and the unlabeled HeLa cells in G1 period. (X1600.) (A) Cell containing the nucleus of HeLa on one side and a bunch of darkly stained mitotic chromosomes of V79-8 (arrow) on the other. In this cell, the HeLa nucleus did not undergo premature chromosome condensation. (B) Autoradiograph of the same cell shown in A. Silver grains (black spots) can be seen on both the mitotic chromosomes and the interphase nucleus. (C) Cell containing highly condensed and darkly stained mitotic chromosomes of V79-8 and the lightly stained PCC of the HeLa cell (arrow). (D) Autoradiograph of the same cell shown in C. Label can be seen on both the mitotic and the prematurely condensed chromosomes.

to be compared are: (i) the chromosome condensation, (ii) the condensed at the time of DNA replication in early S phase. Soon levels of inducers of DNA synthesis during the cell cycle, and after the completion of DNA synthesis, the chromosomes re- (iii) the levels of the chromosome condensation factors. condense progressively through the duration of the G2 period Chromosome Condensation Cycle. The suggestion for the (14) and once again reach the height of their condensation existence of chromosome condensation cycle in mammalian during mitosis (Fig. 4A). It is presumed that the degree of cells made by Mazia (11) was subsequently supported by ex- chromosome condensation that occurs during the G2 period is perimental evidence derived by the use of a variety of tech- of a different order of magnitude than that taking place during niques (9, 12-16). These studies revealed that the chromosomes the initiation of mitosis. In contrast, the chromosomes of V79-8 achieve the maximum degree of condensation during meta- cells decondense rather rapidly. Our data indicate that the phase, gradually decondense during GI, and become least highly condensed metaphase chromosomes of V79-8 cells reach Downloaded by guest on September 30, 2021 5046 Cell Biology: Rao et al. Proc. Natl. Acad. Sci. USA 75 (1978)

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0 1 2 3 Hours after fusion FIG. 3. Frequency of induction of DNA synthesis and premature chromosome condensation in G1 phase HeLa cells after fusion with Time mitotic V79-8 cells. 0, Frequency of PCC induction among the bi- FIG. 4. An idealized diagram to show the differences between nucleate cells, consisting of a set of mitotic chromosomes of V79-8 and HeLa and V79-8 cells regarding some cell cycle parameters. Chro- an interphase nucleus of HeLa; 0, labeling index on either nuclei or mosome condensation cycle of (A) HeLa and (B) V79-8; the process PCC of HeLa among the mitotic-interphase fused cells; A, labeling of decondensation is gradual in HeLa, whereas it is very abrupt in the index among the mono- or the binucleate HeLa cells. case of V79-8 cells. Levels of inducers of DNA synthesis during the cell cycle of (C) HeLa and (D) V79-8 cells; the broken line indicates the critical level. Levels of the mitotic factors during the cell.cycle of an extended state of the early S phase within 30 min (Fig. 1A). (E) HeLa and (F) V79-8 cells; the broken line indicates the critical The condensation process following DNA replication also ap- level. pears to be quite rapid, because we did not find many cells with G2-PCC. The chromosome condensation cycle of V79-8 is contrasted with that of HeLa in Fig. 4. cell enters mitosis when the mitotic factors reach a critical level Levels of Inducers of DNA Synthesis during the Cell Cycle. (Fig. 4E). However, in the case of V79-8 cells, the accumulation The levels of inducers of DNA synthesis are known to fluctuate of mitotic factors must take place during the S phase because during the HeLa cell cycle (Fig. 4C). These inducers remain it has no significant G2 period (Fig. 4F). above the critical level only during the S phase, when they can On the basis of these data, we suggest that the presence or induce DNA in a nucleus upon synthesis G1 fusion between S absence of a Gl period in the cell cycle depends on the levels and G1 cells (5). Mitotic cells of which contain the in- V79-8, of the inducers of DNA synthesis in a cell at the time of mitosis. ducers of DNA synthesis, are capable of inducing both DNA If the inducers are present above a critical level during mitosis, and premature chromosome condensation in synthesis G1 phase the cell enters S phase immediately after cell division. If not, HeLa cells within an hour after fusion (Fig. 3). However, CHO the cell requires a GI period to synthesize the inducers. Thus, cells blocked in mitosis over a of 7-8 hr were unable to period the is induce DNA synthesis in the nuclei of G1 phase HeLa cells duration of GI period directly related to the time required following fusion. This observation rules out the notion that, by the cell to achieve a critical concentration of the inducers. during the extended Colcemid block, the cytoplasm of CHO The slower the synthesis of these inducers, the longer the GI or V79-8 cells advances into S phase while the chromosomes are period. locked in metaphase. Thus it is clear that inducers of DNA synthesis are present and remain above the critical level in the This was Public mitotic cells of V79-8 but not in those of CHO. In spite of the investigation supported by U.S. Health Service Grants CA-16480, CA-11520, CA-19856, and CA-14528 from the presence of the inducers, the metaphase chromosomes of V79-8 National Cancer Institute and GM-23252 from the Institute of General cells did not incorporate label during a 2-3 hr incubation of Medical Sciences. Colcemid-arrested mitotic cells with [3H]thymidine. In earlier studies Rao and Johnson (3) have shown that the chromatin of G2 cells (and probably of metaphase chromosomes) is not 1. Gurdon, J. B. (1967) Proc. Natl. Acad. Sci. USA 58,545-552. available for replication until the chromatids are separated. 2. de Terra, N. (1967) Proc. Natl. Acad. Sci. USA 57,607-614. Levels of the Mitotic Factors. Our earlier studies involving 3. Rao, P. N. & Johnson, R. T. (1970) Nature (London) 225, fusion of early, middle, and late G2 cells revealed that the 159-164. factors for the initiation of mitosis in HeLa cells accumulate 4. Graves, J. A. M. (1972) Exp. Cell Res. 72,393-403. during the G2 period (17). The closer the cells are to mitosis, 5. Rao, P. N., Sunkara, P. S. & Wilson, B. A. (1977) Proc. Natl. Acad. the greater is the level of the mitotic factors they contain. The Sci. USA 74, 2869-2873. Downloaded by guest on September 30, 2021 Cell Biology: Rao et al. Proc. NatI. Acad. Sci. USA 75 (1978) 5047

6. Liskay, R. M. (1977) Proc. Natl. Acad. Sci. USA 74, 1622- 12. Pederson, T. (1972) Proc. Natl. Acad. Sci. USA 69, 2 2224- 1625. 2228. 7. Rao, P. N. & Engelberg, J. (1965) Science 148, 1092-1094. 13. Pederson, T. & Robbins, E. (1972) J. Cell. Biol. 55, 322-327. 8. Rao, P. N. (1968) Science 160, 774-776. 14. Sperling, K. & Rao, P. N. (1974) Chromosome 45, 121-131. 9. Rao, P. N., Wilson, B. A. & Puck, T. T. (1977) J. Cell Physiol. 91, 15. Schor, S. L., Johnson, R. T. & Waldren, C. A. (1975) J. Cell Sci. 131-142. 17,539-565. 10. Wolpert-Defilippes, M. K., Adamson, R. H., Cysk, R. L. & Johns, 16. Hittelman, W. N. & Rao, P. N. (1978) J. Cell Physiol. 95,333- D. G. (1975) Biochem. Pharmacol. 24,751-754. 342. 11. Mazia, D. (1963) J. Cell Comp. Physiol. 62, Suppl. 1, 123- 17. Rao, P. N., Hittelman, W. N. & Wilson, B. A. (1975) Exp. Cell 140. Res. 90, 40-46. Downloaded by guest on September 30, 2021