Journal of Cell Science 105, 113-122 (1993) 113 Printed in Great Britain © The Company of Biologists Limited 1993

Inhibition of transcription blocks progression of NIH3T3

fibroblasts specifically in G1

Sabine Adolph, Sabine Brüsselbach and Rolf Müller* Institut für Molekularbiologie und Tumorforschung (IMT), Philipps-Universität Marburg, Emil-Mannkopff-Strasse 2, D-3550 Marburg, Germany *Author for correspondence

SUMMARY

We have analysed the role of RNA polymerase II-depen- genes thought to be normally induced during S/G2 is not dent transcription in cell cycle progression. Time-lapse required for the completion of an ongoing cell cycle. video recording and cytogenetic analysis were used to S/G2 progression was however clearly dependent on determine the sensitivity of NIH3T3 cells to the RNA protein synthesis. This suggests that cells exposed to - polymerase II inhibitor -amanitin at different stages of amanitin are able to complete their cell cycle because the cell cycle. Our results show that -amanitin blocks sufficiently high levels of mRNA are present in S/G2 due cells specifically in G1, irrespective of the concentration to basal level transcription, or are left from preceding within the range of 3 to 30 g/ml. This indicates that cell cycles. It is therefore unlikely that transcriptional transcription in G1 is required to overcome a restriction regulation in S or G2 plays a crucial role in the control point located in this phase of the cell cycle. In agree- of cell cycle progression in NIH3T3 cells. ment with this conclusion is the requirement for an uninhibited protein synthesis during G1 progression. In addition, the insensitivity of S-phase cells to RNA poly- Key words: cell cycle progression, a -amanitin, transcription, gene merase II inhibition suggests that the transcription of expression,

INTRODUCTION progression past the R point (Campisi et al., 1982; Pardee, 1989). Although candidate ‘R proteins’ have been identi- When mitogens are withdrawn from cells or protein syn- fied (Croy and Pardee, 1983; Pardee, 1989), their function thesis is partially blocked by an inhibitor of translation, they in cell cycle progression remains unknown. It is tempting will arrest in a state of quiescence (G0) or in the G1-phase to speculate, however, that the R point may be under the of the cell cycle, unless they have reached a restriction point control of the tumor suppressor gene product pRB, whose close to the beginning of S-phase, referred to as the R point negative regulatory function is abrogated by phosphoryla- (Pardee, 1974; for reviews see Baserga, 1985; Norbury and tion events occurring in late G1, and/or by the recently iden- Nurse, 1992; Pardee, 1989; Soprano and Consenza, 1992). tified G1-specific and cdc2-related protein kinases This point has been localised in different systems anywhere (for reviews see Hamel et al., 1992; Weinberg, 1991). The between 12 min and 3 h before S-phase entry, and its vari- R point in mammalian cells may thus be analogous to the ability has been suggested to be largely responsible for the START point in S. cerevisiae not only because of its cen- variations in cell cycle length seen in individual cells tral role in cell cycle progression, but also with respect to (Pardee, 1974; Rubin and Steiner, 1975; Yen and Pardee, the underlying molecular control mechanisms (for reviews 1978). Beyond the R point cells no longer require mitogens see Herskowitz et al., 1991; Nasmyth et al., 1991). and are less sensitive to protein synthesis inhibitors, sug- Kinetic analyses performed by time-lapse cinematogra- gesting a key role for this control point in mammalian cell phy of NIH3T3 cells have led to conclusions that can easily cycle progression. This conclusion is in agreement with the be reconciled with the R point model described above observation that the R point is not, or only poorly, func- (Zetterberg and Larsson, 1985). In these experiments, the tional in transformed cells (Medrano and Pardee, 1980; position of cells at different points between and S- Pardee, 1974). The R point has been postulated to be con- phase was determined and the sensitivity of these cells to trolled by (a) labile protein(s) with a half-life of 2-3 h, serum deprivation and the inhibition of protein synthesis called the ‘R protein’, which is synthesised earlier in G1 was analysed (by following the cells to the next mitosis). and whose expression at a critical level is a prerequisite for These experiments clearly showed that cells younger than 114 S. Adolph, S. Brüsselbach and R. Müller

3.5 h were highly dependent on both the presence of mito- MATERIALS AND METHODS gens and maximal protein synthesis, and that the length of Materials this period of G1 was more or less constant. The authors termed this phase the post-mitotic or G1pm-phase. The cells Cycloheximide and a -amanitin were obtained from Sigma. The antibody for BrdU detection was purchased from Partec and used then entered the presynthetic or G1ps-phase, which was independent of serum, but highly variable in length; some at a 1:500 dilution. The antibody to detect c-Fos protein has been described elsewhere (Verrier et al., 1986) and was used at a 1:30 cells entering S-phase almost immediately after leaving dilution. The second antibodies for indirect immunohistochemical G1pm, others remaining in G1ps for up to 10 h. It is very staining were either a Cy3- or a HRP-coupled rabbit anti-mouse likely that the R point discussed above defines the switch IgG (Dianova). from G1pm to G1ps. Very similar observations were also made with human diploid fibroblasts (Larsson et al., 1989), Time-lapse video recording (TLV) showing that the described behaviour is not a peculiarity of NIH3T3 cells were cultured under standard condition (5% CO2, NIH3T3 cells. 37°C) in DMEM with 10% FCS. For video recording, about 105 Upon mitogen stimulation of quiescent fibroblasts a large cells were seeded into a 25 cm2 plastic dish, cultured for one day number of genes are transcriptionally activated (for reviews in a CO2 incubator and then transferred to an inverted microscope see Bravo, 1990; Hershman, 1991). The diversity of the (Zeiss, Axiovert 35) equipped with a temperature- and CO2-con- genomic response to mitogen stimulation is underscored by trolled stage. Cell proliferation was recorded by a CCD camera (CF 15 MC, Kappa) coupled to a video recorder (AG 7350, Pana- the fact that genes associated with many different cellular sonic) for 40 to 60 h. After observing the cells for 18 to 24 h, the processes are activated, including secreted factors, proteins inhibitors (a -amanitin or cycloheximide) were applied and cells involved in building or degrading the extracellular matrix, were recorded for another 24 to 30 h. At least 100 individual cells metabolic enzymes and other proteins associated with per film were followed from mitosis to mitosis and the intermi- energy metabolism and transport processes, enzymes totic times (tc) were determined. involved in nucleotide and DNA synthesis, proteins involved in signal transduction, and transcription factors. In Data analysis and representation addition, a number of cell cycle genes, most notably of the To analyse the distribution of cell cycle lengths of untreated cells cdk and families, have been identified in recent years we chose the method of Shields and Smith (Shields and Smith, (Lew and Reed, 1992; Meyerson et al., 1992). Many of 1977). These authors defined the proportion of cells which have these genes and their products are activated at specific an intermitotic time greater or equal to t as a (t) and plotted loga against the intermitotic time (t ). Since there is a minimum inter- stages of the cell cycle after mitogen stimulation of quies- c mitotic time (TB), no cells will divide in a period less then TB, cent cells or after the release from metabolic blocks. These leading to the corresponding value of a =100%. According to the genes include those encoding the S- and M-phase-specific transition probability model of cell cycle progression, a theoreti- and the mitotic B-type cyclins (Pines and Hunter, cal plot of loga against time would have two components, a lag 1989, 1990), the cdc2 protein kinase (Lee and Nurse, 1987) phase equal to TB, and an exponential phase which will be linear and the protein phosphatase (Sadhu et al., 1990), in the loga plot. This presentation of the data proved especially whose function is indispensable for S and/or G2 progres- useful to compare the tc values of cells hit by the drug during an sion (Riabowol et al., 1989; Walker and Maller, 1991; ongoing cell cycle. In addition, the cells’ age at the time of drug Girard et al., 1991; Hoffmann et al., 1993; for a review on application (tx) as well as the time they needed to finish a proper mitosis after drug application (ty) were listed and plotted against the role of these genes in S. pombe see Forsburg and Nurse, each other. 1991). Very little is known, however, about the transcrip- tion of these genes and their role during cell cycle pro- Cytogenetic techniques gression in normally cycling, non-synchronised mammalian Cells were grown directly on microscope slides in Quadriperm cells. It thus remains unclear which stages of the cell cycle dishes (Heraeus) and chromosomes were prepared in are dependent on the expression of specific sets of genes. situ by the standard technique. To determine the duration of the This is an important issue since it is likely that such genes G2/M-phase, tG∑, bromodeoxyuridine (BrdU; 20 mg/ml) was encode the proteins required to overcome the restriction applied for increasing times to the cell culture (from 1 to 6 h) points in the cell cycle. As a first step in addressing these before chromosome preparation. The incorporated BrdU was open questions we have determined, by time-lapse video detected by the anti-BrdU antibody technique as described (Vogel et al., 1986). The frequency of unlabelled metaphase plates gives recording and cytogenetic analysis of bromodeoxyuridine- the frequency of cells which were in G at the time of BrdU appli- substituted chromosomes, the sensitivity of NIH3T3 cells 2 cation. To determine the duration of G2/M and S, tS+G∑, BrdU was at different stages of the cell cycle to the RNA polymerase applied for increasing times to the cell cultures from 7 to 15 h II inhibitor a -amanitin (Wieland and Faulstich, 1991). We before chromosome preparation. The incorporated BrdU was visu- find that only the G1-phase is sensitive to inhibition by a - alised by fluorescence microscopy either directly by acridine amanitin, which is in agreement with the observation that orange staining (Dutrillaux et al., 1973) or by Hoechst staining G1 is also the most sensitive phase with respect to the inhi- (Latt, 1973). Cells which incorporated BrdU over their total length bition of protein biosynthesis by cycloheximide. Our results of S will have pale, weakly stained chromosomes due to quench- also suggest that an unimpaired transcription of many cell ing of fluorescence emission by the incorporated BrdU. Those cells which started to replicate their DNA before the application cycle genes normally induced during S/G2 is not required of BrdU will show brilliant, fluorescing bands on their chromo- for the completion of an ongoing cell cycle. These findings somes. The frequency of metaphase plates with completely pale place transcriptionally controlled restriction points pre- chromosomes corresponds to the frequency of G1 cells at the time dominantly or even exclusively in G1. of BrdU application. The proliferation of cells in the presence of Restriction of cell cycle progression 115 the inhibitor was also analysed by replication analysis of Table 1. Cell cycle parameters of untreated NIH-3T3 metaphase chromosomes. Cells were seeded onto slides in cells Quadriperm dishes and labelled for the first S-phase with BrdU Frequency tG∑/M tS+G∑/M tS tGœ (25 h). Thereafter, medium was changed and supplemented with tc (h) (%)* (h)† (h)† (h)‡ (h)‡ thymidine (10 mM) and the inhibitors. About 2 days later, chro- ³ 10 97 1.7-2.4 7.0-8.2 5.3-5.8 1.8-3.0 mosomes were harvested in situ as described above and the incor- ³ 11 90 2.0-2.7 7.2-9.0 5.2-6.3 2.0-3.8 porated BrdU was detected by the antibody technique. The seg- ³ 12 80 2.4-3.4 7.8-9.2 5.4-5.8 2.8-4.2 regation of the original BrdU-substituted chromatids in the ³ 13 62 2.7-3.5 8.1-9.6 5.4-6.1 3.4-4.9 thymidine makes it possible to calculate the number of cycles (S- ³ 14 45 3.2-4.0 8.8-10.0 5.6-6.0 4.0-5.2 phases) the cells completed during drug application. ³ 15 28 3.6-5.2 9.4-10.8 5.8-5.6 4.2-5.6 ³ 16 20 3.8-5.8 10.0-11.2 6.2-5.4 4.8-6.0 Measurement of protein biosynthesis ³ 18 10 4.2-7.0 11.8-12.2 7.6-5.2 5.8-6.2 ³ 20 5 4.9-8.0 12.2-13.0 7.3-5.0 7.0-7.8 Cells were pulse-labelled for 2 h in medium containing ³ 30 2 5.7-8.7 12.8-13.5 7.1-4.8 ³ 7.0 [3H]isoleucine (NEN, 2 mCi/ml), 10% of the normal concentra- tion of unlabelled isoleucine and different concentrations of cyclo- *Determined by time-lapse analysis for tc and by cytogenetic analysis heximide. The incorporated [3H]isoleucine was determined in the for tG∑/M or tS+G∑/M. acid-insoluble material by placing it in scintillation fluid and †Determined by cytogenetic analysis of metaphase chromosomes after counting in a Beckman scintillation counter. Radioactivity of BrdU exposure. untreated cells was taken as 100%. Values from several experiments were combined. ‡Calculated as: tS=tS+G∑/M- tG∑/M and tGœ=tc- tS+G∑/M. Measurement of mRNA synthesis The inhibition of RNA polymerase II transcription by a -amanitin was analysed by c-Fos induction in newly stimulated 3T3 cells cytogenetic analysis gives the tG∑/M and tS+G∑/M values with- after serum starvation. At various times before stimulation, serum- out reference to the corresponding tc values. The duration starved cells were treated with a -amanitin (10 mg/ml), and 1 h of G1 (tGœ) can only be estimated as tc- tS+G∑/M. The two after serum stimulation the level of Fos protein in individual cells data sets were therefore combined assuming that t values was determined immunohistochemically (Verrier et al., 1986) and c quantitated as described (Quantimet, Leica; Lucibello et al., 1993). and the lengths of cell cycle phases correlate, i.e. that the cells with the shortest tc also have the shortest tG∑ and tS+G∑ values, and conversely, that long tc, tG∑ and tS+G∑ values RESULTS are also linked. Table 1 summarises the tc, tG∑ and tS+G∑ Cell cycle parameters of untreated cells values measured as described above and the corresponding calculated tS and tGœ values. This way the average tS and We first determined the intermitotic times (tc) of more than tGœ values were found to be 6 h and 4.5 h, respectively. The 400 untreated cells from several experiments by time-lapse minimal time for G1 can be calculated to be 2 h. video recording (TLV) to establish the normal distribution One goal of our investigation was to inhibit RNA poly- of the cell cycle lengths in the population of NIH3T3 cells merase II transcription by a -amanitin and to identify phases used in the present study. The shortest tc determined was in the cell cycle that differ in their sensitivity to the drug. 8.5 h (in 0.5% of the cells). Half of the population needed Therefore, our interest focused on those cells that were hit 13 to 14 h to complete a cell cycle, and 90% of the cells during an ongoing cell cycle. The age of a given cell at the showed tc values of 10 to 18 h. Only 5% had a cell cycle time of drug application (tx) as well as the time that cell longer than 21 h, the longest found being 34 h. needed after drug application to complete the cell cycle (ty) After culturing the cells for different times in BrdU, cyto- were determined by TLV. In addition, we sought to estab- genetic analysis of the metaphase plates enabled us to deter- lish a technique for assigning a given tx value to a cell cycle mine the frequency of BrdU-unlabelled metaphase plates. phase. For this purpose, tx and ty values were plotted against In addition, we established the fraction of cells that repli- each other. Fig. 1 shows such a typical tx/ty-plot for mock- cated their DNA in BrdU over the total length of S-phase. treated cells. Each line represents cells showing the same These two frequencies correspond to the duration of G2/M cell cycle length tc=tx+ty, hit at a cell age of x hours and (tG∑/M) and S+G2/M (tS+G∑/M), respectively. In several completing their cell cycle y hours after inhibitor applica- experiments, about 100 metaphase plates were analysed for tion. The shaded area covers the expected tc values from each BrdU exposure. These results are presented in Table 10 to 18 h, corresponding to 90% of untreated cells. Since 1. The shortest tG∑/M was 1.7 h. After 3.5 h exposure, 50% ty also corresponds to the time of BrdU application in the of the metaphase plates were BrdU-labelled, and after 8 h cytogenetic analysis it is possible to include the distribu- the corresponding value was 95%. The shortest tS+G∑/M was tion of G2, S, and G1 in this plot. We incorporated tG∑/M 7 h, and no cell, out of 200 , needed longer than and tS+G∑/M from Table 1 as ty values into this plot and cal- 14 h to complete S+G2/M. Nine to ten hours were neces- culated the corresponding tx values as tc- ty. This enabled sary for 50% of the cells to pass through S+G2/M. There- us to estimate the position of individual cells in the cell fore, the average value for tG∑/M is 3.5 h and for tS+G∑/M cycle at the time of drug application. Any cell dividing in 9.5 h. the left-most field (left of the upper dotted line) was likely Both methods, TLV and the cytogenetic analysis, are to be in G1 at the time of the treatment. Any cell dividing used to study individual cells. The former technique yields in the field between the dotted lines was likely to be in S, exact tc values without any information about the duration and any cell dividing in the right-most field (right of the of the different cell cycle phases; on the other hand, the lower dotted line) should have been in G2 at the time of 116 S. Adolph, S. Brüsselbach and R. Müller

Fig. 1. Typical tx/ty-plot of mock-treated cells. tx values were determined by TLV and are equivalent to the cell age at treatment. The ty values represent the time cells needed to complete the cell cycle after drug application as determined by TLV. In addition, ty values correspond to the time of BrdU exposure prior to cytogenetic analysis. The total cell cycle length of a given cell is equal to tc=tx+ty. Cells with the same cell cycle length (tc) are represented by lines. To determine the borders of G1/S and S/G2 the values of tS+G∑/M (᭿) and tG∑/M (+) from Table 1 were included as ty values. The corresponding tx values were calculated by correlating the frequency of tc values with the frequencies of tS+G∑/M and tG∑/M. The broad dotted lines represent the approximate border of G1/S and S/G2. The shaded field marks the area of tc values from 10 to 18 h, corresponding to 90% untreated cells. Fig. 2. Inhibition of c-Fos induction by a -amanitin and total protein synthesis by cycloheximide. (a) Serum-deprived NIH3T3 drug application. The validity of this interpretation was con- cells were stimulated with 10% FCS after different times of a - firmed by experiments where the same cells observed by amanitin pretreatment (10 mg/ml). Cells were fixed (‘harvest’) 1 h TLV were analysed for incorporation of BrdU (data not after stimulation and stained for Fos expression by indirect shown; also see Fig. 7). A cell with the same age at treat- immunofluorescence. The fluorescence intensity in 300 individual cells was measured by digital image analysis (Quantimet, Leica) ment but a different ty value can either have a normal but exceptionally high t , or might have a prolonged t value and average fluorescence levels were plotted against time. c y (b) NIH3T3 cells growing in DMEM plus 10% FCS were caused by the drug. Since it is impossible to decide this by metabolically labelled with [3H]isoleucine in the presence of analysing single cells, we used the following definition to different concentrations of cycloheximide. The radioactivity in identify a sensitive cell cycle phase: none, or only a few acid-precipitated material was determined and compared to the cells of the same age at treatment, divide in the expected incorporation of [3H]isoleucine into cells in the absence of field of the tx/ty-plot, and the fraction of cells that reach the cycloheximide (100%). end of recording without division is significantly increased.

Influence of -amanitin and cycloheximide on NIH3T3 cells. Addition of 10.0 mg/ml a -amanitin 3 h prior proliferation, transcription and protein to serum stimulation was sufficient to block fos expression biosynthesis by ³ 90% and half maximum inhibition was seen after about To determine the kinetics of a -amanitin on RNA poly- 80 min. Evaluation of the immunofluorescence analysis also merase II-dependent transcription in NIH3T3 cells, we showed that a -amanitin affected c-fos expression in all cells measured the level of c-Fos in individual cells one hour of the population to a similar extent (data not shown). This after stimulation with serum following different times of a - suggests that cells dividing after ³ 2 h of exposure to the amanitin pretreatment (Fig. 2a). The c-fos gene was chosen drug should also be affected in their RNA polymerase II because its transcription is rapidly induced by mitogens and transcription. its mRNA is also rapidly translated: maximum protein We then investigated the influence of a -amanitin on the levels are reached 1 hour after serum stimulation of proliferation of NIH3T3 cells to be able to choose appro- Restriction of cell cycle progression 117

Table 2. Proliferation of cells after treatment with - 100 amanitin and cycloheximide for 2 days Fraction of cells that passed through n number of S-phases after drug application 50 Treatment Number of (concn in mg/ml) n³ 4 n=3 n=2 n=1 n=0 metaphases Untreated 48 36 16 0 0 185 a -Amanitin 1.0 2 21 64 4 0 55 20 3.0 0 7 36 57 0 14 9.0 0 0 0 0 100 <5 15.0 0 0 0 0 0 none CHX1.0 Cycloheximide 10 0.01 13 47 37 10 0 136 0.05 0 23 47 30 0 91 0.1 0 0 0 100 0 14 0.5 0 0 0 0 100 <5 5 1.0 0 0 0 0 0 none

Freshly seeded cells were pulse-labelled for the first S-phase (25 h) with = Cumulative fraction of cells (%) BrdU, then the medium was changed and supplemented with thymidine plus either a -amanitin or cycloheximide at the concentrations indicated 2 in the Table. Two days later (51 h for a -amanitin and 43 h for cycloheximide, respectively) metaphase chromosomes were prepared and C the incorporated BrdU was detected immunohistochemically. Segregation ama30 CHX0.02 of the BrdU-labelled chromatids allowed us to calculate the number of 1 replication cycles in thymidine. 0 5 10 15 20 25 30 35 40 45 50

tc (h)

Fig. 3. Distribution of tc values of cells exposed to cycloheximide priate concentrations of the drugs for subsequent experi- or a -amanitin. Only those cells were included that started their ments. The proliferation of cells in the presence of a -aman- cell cycle before the start of treatment. The fraction of cells (a ) itin as analysed by the cytogenetic approach clearly with a tc value equal to or bigger than a given value (on the depended on the concentration used (Table 2). Almost half ordinate) is plotted as loga against tc. Since there is a minimum of the untreated cells replicated at least 4 times in 51 h, intermitotic time (TB), no cells will divide in a period less then TB and 13% progressed through only 2 consecutive S-phases. (marked by vertical lines at the top),giving a value of a =100%. The small dots represent cells that reached the end of recording At a concentration of 1.0 mg/ml a -amanitin most of the without division. C, untreated control cells; CHX 0.02 and CHX cells progressed through only 2 consecutive S-phases and 1.0, cells treated with 0.02 and 1.0 mg/ml cycloheximide, 9.0 mg/ml were sufficient to prevent completion of a sub- respectively; ama30, cells treated with 30 mg/ml a -amanitin. sequent cell cycle. The mitotic index was strongly decreased for concentrations ³ 3.0 mg/ml. No metaphase spreads at all could be found at 15.0 mg/ml, and none of the nuclei (³ 2000) showed incorporation of a visible Influence of -amanitin and cycloheximide on t amount of thymidine during the subsequent S-phase. This c led to the conclusion that 15 mg/ml a -amanitin were suffi- To compare the effect of the drugs on tc of an ongoing cell cient to block entry into the subsequent S-phase. cycle we plotted the distribution of tc as a -curves (Fig. 3) Since one aspect of the present study was to compare the as described in the Materials and Methods. Even the lowest role of protein and RNA synthesis in cell cycle progres- concentration of cycloheximide (0.02 mg/ml, reducing pro- sion, the protein biosynthesis inhibitor cycloheximide was tein synthesis by 50%) resulted in a clear increase in tc. included in all subsequent experiments. The proliferation of This was even more pronounced at 1.0 mg/ml, which led to cells in the presence of cycloheximide was analysed in the a reduction in protein synthesis to 10%. In contrast, all con- same way as described above for a -amanitin. A concen- centrations of a -amanitin (3.0, 6.0, 30.0 mg/ml) gave the tration of 0.01 mg/ml cycloheximide (corresponding to same picture; about 50% showed a tc similar to cells treated about 40% reduction of protein biosynthesis; Fig. 2b) had with 0.02 mg/ml cycloheximide, whereas the other 50% only a marginal effect on cell proliferation (Table 2). Appli- gave rise to clearly increased tc values. The small dots in cation of 0.05 mg/ml cycloheximide (giving about 60% Fig. 3, which represent cells that reached the end of record- reduction of protein biosynthesis) for 2 days reduced the ing without division, indicate that a large proportion of cells number of consecutive S-phases to 2 and 0.5 mg/ml (reduc- treated with 30 mg/ml a -amanitin had cell cycle times that tion of 85% in protein synthesis) were sufficient to prevent might even be much longer. the completion of any further S-phase during the 2 days of the analysis. The mitotic index decreased clearly after appli- Stage-specific inhibition of cell cycle progression cation of 0.1 mg/ml or more. By these criteria 0.5 and 1.0 by -amanitin and cycloheximide mg/ml cycloheximide have a comparable effect on cell pro- The influence of the cell age at treatment, tx, on the liferation to 9.0 and 15.0 mg/ml a -amanitin, respectively. inhibitory effect of the drug on an ongoing cell cycle was 118 S. Adolph, S. Brüsselbach and R. Müller

40 40 a control a -amanitin, 3 µg/ml

30 30

³ 24 h 20 20 G1

10 10

0 0 0 10 20 0 10 20

40 40 b cycloheximide, 0.02 µg/ml b -amanitin, 6 µg/ml

30 ³ 29 h 30

³ 24 h 20 20 G1

10 10 y = Time until next mitosis (h) t y = Time until next mitosis (h) t 0 0 0 10 20 0 10 20

50 c cycloheximide, 1 µg/ml 40 c -amanitin, 30 µg/ml ³ 42 h 40 30 ³ 24 h 30

20 20 G1

10 G1+S 10

0 0 0 10 20 0 10 20 t x = Cell age at treatment (h) t x = Cell age at treatment (h)

Fig. 4. Relationship between cell age (tx) and the time (ty) Fig. 5. Relationship between cell age (tx) and the time (ty) required to complete the cell cycle after cycloheximide required to complete the cell cycle after a -amanitin application. application. (a) Untreated control cells; (b) cells treated with 0.02 (a) 3 mg a -amanitin/ml; (b) 6 mg a -amanitin/ml; (c) 30 mg a - mg cycloheximide/ml; (c) cells treated with 1 mg amanitin/ml. For details see legend to Fig. 4. cycloheximide/ml. Each solid square represents a dividing cell that was hit at the age of x hours and needed y hours to complete its cell cycle. The open squares represent cells that completed determined by TLV. The results are presented in Figs 4 and their cell cycle before treatment. The shaded field marks the area 5 as tx/ty plots, as described above. Untreated control cells of tc values from 10 to 20 h, i.e. where ³ 90% of the untreated cells divided in the expected area (compare Fig. 1) with only a are found. The boxed area indicates cells that reached the end of few cells showing tc values >20 h (Fig. 4a). The behaviour recording without division. The area expected for cells that were of cells treated with a concentration of cycloheximide that in G1 or S at the time of exposure to the drug is marked in (c). reduced protein synthesis to about 50% (0.02 mg/ml) is shown in Fig. 4b. Many of the cells divided in the area expected for untreated cells, but a number of cells showed an increased ty, and thus tc. Increasing the cycloheximide Restriction of cell cycle progression 119

Fig. 7. Simultaneous incorporation of BrdU and TLV. The Fig. 6. Fraction of non-dividing cells in different age groups. The experiment was carried out as described in Figs 4 and 5, except percentage of a -amanitin treated cells that reached the end of that the cells were exposed to BrdU for 1 h directly before recording without dividing is plotted against the cell age at exposure to a -amanitin. Incorporation of BrdU was determined treatment. Each group of cells comprises a 2-hour interval. The after recording by immunostaining. Solid squares represent BrdU- three experiments shown in Fig. 5 were combined for the plot labelled cells, open squares are unlabelled cells. Gray lines shown above. indicate the approximate positions of the borders between the G1/S and S/G2 compartments, as explained in Fig. 1. to a concentration where 90% of the protein biosynthesis was inhibited caused a complete depletion of cells in the G1 and S fields; all these cells reached the end of the video the 10th cell, hit at the age of approx. 14 h, could not, how- without dividing (Fig. 4c). Some of the cells with a cell age ever, be determined by this approach. Similar findings were at treatment of 9 to 14 h showed a significantly prolonged made in a number of other experiments. In total, 61 cells total cell cycle time. Other cells of the same age class either were counted that reached the end of recording without did not divide at all or completed their cell cycle with a division; 56 out of these cells were younger than 8 h. These proper mitosis in the expected time. Such cells were prob- observations strongly support our conclusion that G1 is by ably in G2 at the time of treatment. far the most sensitive phase with respect to the action of The three experiments with a -amanitin (3.0, 6.0, 30.0 a -amanitin. In addition, the data in Fig. 7 indicate a normal- mg/ml) gave very different tx/ty plots when compared to sized pool of S-phase cells, suggesting that a -amanitin cycloheximide. In addition, the results were largely inde- treatment did not lead to a prolongation of S/G2 progres- pendent of the concentration used (Fig. 5). At all concen- sion. trations many cells divided in the expected area of the tx/ty Finally, we wished to confirm the insensitivity of S/G2 plots. Only the population of cells at the age of 0 to 2 h cells to the inhibitory effect of a -amanitin by a second, was highly affected, pointing to a block in G1. In agree- independent, approach. We determined, by the analysis of ment with this conclusion, none of the cells shown in Fig. metaphase chromosomes, the effect of a -amanitin and 5 divided a second time in the presence of the drug. cycloheximide on the progression through S and G2. Apply- A number of cells that were older than 2 h at the time ing BrdU for different intervals before chromosome prepa- of treatment reached the end of the video without dividing ration allowed us to determine the frequencies of cells in (Fig. 6). From the TLV data obtained so far it was not pos- S or G2 at the time of BrdU addition. Table 3 summarises sible to decide in which cell cycle phase these cells were these results. a -Amanitin had no effect on the frequencies hit. To answer this question we analysed, by TLV, cells of S and G2 cells. The small differences seen in Table 3 that were pulse-labelled for one hour with BrdU before the clearly fall within the range of experimental variation. On addition of a -amanitin (10.0 mg/ml). After recording, the the other hand, even a low concentration of cycloheximide incorporated BrdU was detected immunohistochemically. (0.05 mg/ml) led to a decrease in the number of S-phase All cells that reached the end of recording without divid- cells, and at the higher cycloheximide concentration (0.1 ing were not labelled by BrdU (Fig. 7), which means that mg/ml) the pool of G2 cells was also clearly enlarged. These these cells were either in G1 or in G2. However, 9 out of independent experiments demonstrate again that inhibition 10 cells (boxed area in Fig. 7) were younger than the min- of protein biosynthesis by approximately 50% already has imum time required for progression through G1 and S a prolonging effect on S/G2. In contrast, even a high a - (approx. 8 h; see Table 1). Therefore, these cells were in amanitin concentration showed no significant effect on the G1 when exposed to BrdU and a -amanitin. The stage of progression through S and G2. 120 S. Adolph, S. Brüsselbach and R. Müller

Table 3. Influence of -amanitin and cycloheximide on depleted of cells younger than 2 h, and that the fraction of the frequencies of S and G2 cells as determined by undivided cells dramatically increased (boxed areas in Fig. cytogenetic analysis of metaphase chromosomes 5). This is different from the effects seen with cyclohex- Number of imide, which at a low concentration also showed an effect Treatment BrdU S-phase of exp. on young cells (<5 h), but at higher concentrations also led (mg/ml) (h) (h) G GL GLL G2-phase metaphases to a dramatic prolongation and block of cell cycle pro- Untreated gression in older cells. 2 - - 2 98 7 826 The next important task was then to determine in which 3 - 4 23 73 7 788 cell cycle phases the inhibitors exerted their effects, i.e. to 4 7 24 44 24 10 899 5 13 35 31 16 5 969 relate the cell’s age to its position in the cycle. 6 33 35 26 8 6 395 For this purpose we combined the results obtained by two a -Amanitin different techniques, the TLV, which allowed for the pre- 10.0 3 3 - 1 29 70 1 109 cise measurement of total cell cycle times, and a cytoge- 4 4 6 27 33 34 1 110 netic approach, which gave us accurate information about 5 5 10 44 22 24 1 123 the length of S+G2 and G2. Based on the assumption that Cycloheximide 0.05 6 5 - 23 34 43 1 69 a prolonged cell cycle time can be correlated with a longer 6 6 3 33 40 23 1 106 duration of the different cell cycle phases we established 0.1 6 5 - 1 23 77 1 151 the data evaluation plot shown in Fig. 1. The validity of 6 6 - 7 26 67 1 134 this technique for relating cell age and the position in the Cells were seeded on slides and incubated with BrdU and the drugs as cell cycle was tested by identifying S-phase cells through indicated. BrdU incorporation was analysed by the antibody technique. BrdU labelling within a cell population followed by TLV Unlabelled chromosomes indicated that the cells were in G2 at the time of (Fig. 7 and data not shown). In the experiment shown in BrdU application. Labelled cells were grouped according to their Fig. 7, 16 cells were BrdU-labelled and 14 out of these chromosomal replication banding pattern in three groups. G, GL and GLL were found in the predicted area. Conversely, 31 cells represent normal, late and very late G-bands, respectively. Normal G- bands correspond to the standard idiogramme of G-banded chromosomes remained unlabelled and 29 out of these appeared in the (Evans, 1989). correct area of the plot in Fig. 7. Similar results were obtained in other experiments (data not shown). The frac- tion of ‘incorrectly’ assigned cells was therefore 2/16 (12.5%) and 2/31 (6.4%), respectively, i.e. there are some DISCUSSION cells with a long G1 and a short S/G2-phase The value of approximately 90% correctly positioned cells can be con- This study was undertaken to investigate the role of tran- scriptional regulation in the control of cell cycle progres- sidered sufficiently high to allow a conclusive interpreta- sion. To this end, we have performed time-lapse video tion of the data. On this basis we were able to assign cell recording (TLV) and cytogenetic analyses to determine the cycle phases to the effects seen in Fig. 5; a -amanitin has sensitivity of NIH3T3 cells at different stages in the cell a dramatic effect on cells in early G1 (£2 h post-mitosis), cycle to the RNA polymerase II inhibitor a -amanitin. The but clearly also affects cells at later stages in G1, while cells results of this study were also compared to observations in S and G2 are largely resistant to the inhibitory effect of made with the protein biosynthesis inhibitor cycloheximide a -amanitin. This is supported by the data in Fig. 6, where in analogous experiments. The most intriguing finding of the number of cells blocked in cell cycle progression by a - amanitin was plotted against the cell age. These data also this study is the observation that G1 is highly sensitive to both types of inhibitors, while S and G2 progression is show the sensitivity of the cells to a -amanitin within the blocked to a significant extent only by higher concentra- first 8 h post-mitosis, in that >90% of the cells that did not tions of cycloheximide. divide until the end of recording were 8 h or younger. In the first part of this investigation, we determined the However, the conclusive interpretation of the data effect of a -amanitin and cycloheximide on intermitotic required the clarification of another question, i.e. the kinet- times, i.e. the total cell cycle lengths (tc). The results of ics of a -amanitin action and a quantitative assessment of these experiments are shown in Fig. 3. It is obvious that in its effect on RNA polymerase II-dependent transcription. both cases the fraction of cells with higher tc values was Unfortunately, such a measurement is not as easy as in the increased. In the case of a -amanitin, at least 3 different case of protein biosynthesis inhibitors, because radioactive populations of cells were detectable: cells with largely RNA precursors are not exclusively incorporated into normal tc values (top part of the curve); cells with greatly mRNA by RNA polymerase II, but to much higher extent increased intermitotic times; and cells that had not divided into rRNA and tRNA by the other RNA polymerases. We by the end of recording. The important question of where therefore decided to measure the effect of a -amanitin on in the cell cycle these cells were blocked was analysed in the transcription of a single gene. This was possible, since the experiment shown in Fig. 5 (compare to control in Fig. a -amanitin should affect the transcription of all genes to a 4a). Here, the influence of cell age on the inhibitory effect similar extent due to its inhibition of RNA polymerase II of a -amanitin was studied. The data indicate that the major- rather than of a gene specific factor. For this purpose we ity of the cells at an age of <8 h were clearly affected, par- chose the c-fos gene whose transcription is induced within ticularly those cells at £2 h post-mitosis. This is evident minutes after mitogen stimulation. In addition, the c-fos from the fact that the respective area in the plots was gene product, c-Fos, is rapidly synthesised as well, so that Restriction of cell cycle progression 121 gene induction and its inhibition by a -amanitin could be mean that these genes behave in a similar way or even have followed at the single cell level. The results of this exper- a function during G1 progression in normally cycling cells. iment (Fig. 2a) showed that a -amanitin at a dose of 10 One example of this kind is the c-fos gene, which is induced mg/ml inhibits c-Fos induction by 50% within 80 min. The to very high levels by mitogen stimulation but which is kinetics of a -amanitin mediated inhibition on RNA poly- expressed only at very low levels throughout the cell cycle merase II are therefore fast enough to conclude that the (Bravo et al., 1986) and apparently has no crucial function majority of the cells were in G1 at the time of a -amanitin in normally cycling cells (Kovary and Bravo, 1991, 1992). action. In addition, in none of the experiments we per- One might, however, speculate that genes whose transcrip- formed did we find a single cell in S-phase (identified by tion is not regulated to a significant extent during G1® S BrdU labelling) whose cell cycle progression was visibly progression, but whose transcripts have a short half-life are prolonged or even blocked by a -amanitin. the critical targets for the action of a -amanitin. Among such One of the most surprising results of the present study genes may be the recently identified cyclins C, D and E was the finding that a -amanitin has no detectable effect on (Lew and Reed, 1992), members of the cdk family (Mey- cells in S or G2. This conclusion is based on the evalua- erson et al., 1992) and the transcription factor (Nevins, tion of a large number of cells by TLV (375 cells) and was 1992). Future experiments making use of anti-sense RNA fully confirmed by cytogenetic analyses (Table 3). This approaches or targeted gene disruption by homologous indicates that the transcription of a large number of genes, recombination will have to clarify the relevance of these including many cell cycle genes, can be blocked by >90% genes for the progression of cells through the G1-phase and without any significant effect on the completion of an ongo- their entry into S. ing cell cycle. This is surprising as such genes include, for example, cdc2 and , which have been reported to This work was supported by the Deutsche Forschungsgemein- be induced in late S and G2 (Dalton, 1992; Pines and schaft (Mu601/5-2, Mu601/5-3 and Mu601/7-1) and the Dr Mil- Hunter, 1989). The question thus arises how these cells dred Scheel-Stiftung für Krebsforschung. S.B. is the recipient of manage to complete their cell cycle with a proper mitosis. a fellowship from the Graduiertenkolleg ‘Zell- und Tumor-biolo- gie’ at the Philipps-Universität Marburg. Our own and other authors’ observations have shown that cells need an active protein synthesis machinery to proceed through S and G2 (Zetterberg and Larsson, 1985; Figs 3 REFERENCES and 4). This leads to the conclusion that the a -amanitin treated cells must contain sufficiently high levels of mRNA Baserga, R. (1985). The Biology of Cell Reproduction. Cambridge, to be able to synthesise the required amounts of protein. 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(Received 15 February 1993 - Accepted 10 March 1993)