Eur. J. Biochem. 210, 389-398 (1992) 0FEBS 1992

Reversible shutdown of replicon initiation by transient hypoxia in Ehrlich ascites cells Dependence of initiation on short-lived protein Hans-Jnrg RIEDINGER, Volker GEKELER and Hans PROBST

Physiologisch-chemischeslnstitut der Universitat Tiibingen, Federal Republic of Germany (Received June 26/August 31, 1992) - EJB 92 0888

The 0,-dependent regulation of replication in Ehrlich ascites cells, characterized by a reversible shutdown of replicon initiation during hypoxia, was scrutinized with respect to the involvement of gene expression. Synchronous and asynchronous cells were subjected to transient hypoxia and examined for expression of selected ‘late’ growth-regulated mRNA and for the influence of inhibitors of transcription and translation on DNA replication. Trrespective of whether replicon initiation was suppressed by hypoxia or retriggered by reoxygenation, the levels of thymidine kinase mRNA and of proliferating cell-nuclear antigen/cyclin mRNA were as high as in untreated replicating cells. The level of histone H3.1 mRNA followed, with a distinct delay, the replicative activity of the cells governed by the imposed changes of p 0 2 . The response of replication to inhibition of transcription and translation was virtually the same as to hypoxia, i.e. a selective suppression of replicon initiation. It was demonstrated that replicon initiation depends on one or several short-lived protein(s) (lifetime about 5 min) which is (are) formed under hypoxic conditions as well. The lifetime of the corresponding RNA message(s) is in the range of several hours. It is suggested that the expression of genes conditioning resting cells for DNA replication remains unaffected by hypoxia or by restoring the normal PO,. Hypoxic cells appear to rest in a state fully prepared for entering DNA replication, but a yet unknown event essential for replicon initiation is blocked. This event depends on a critical oxygen tension as well as on short-lived protein(s).

In previous communications, we demonstrated that DNA replication in Ehrlich ascites cells is subject to a regulation which depends on the O2 tension in the cellular environment [l - 41. This regulation operates at 0, tensions distinctly above the minimum required to support mitochondria1 respiration [5] and to establish a normal adenylate energy charge [ 3 ] . Furthermore, we have shown that this regulation takes place during ascites tumor growth in vivo [3, 41. There, it may be a major determinant of tumor-cell propagation by adapting the tumor cell to the supply of nutrients (not only of 0,) which is increasingly impairing when the tumor mass in the peritoneal cavity grows. Work with cultured cells under controlled O2tension [3,4, 61 revealed the responses of the cellular replication machinery. When the PO, is reduced to values of 200 -2000 ppm (relative to l o 5 Pa total pressure), scheduled replicon initiations are specifically, reversibly and coordinately suppressed, whereas DNA chain growth and maturation in replicons already initiated before the reduction of the p 0 , continue essentially normally. Re-elevating the p 0 2triggers, within a few minutes, a burst of initiations. Thereby, no detectable over-replication, i.e. repeated initiations of the same replicons within a single Correspondence to H. Probst, Physiologisch-chemisches Insljtut der Universitat Tubingen, Floppe-Seyler SLraDe 4, W-7400 Tubingen 1, Federal Republic of Germany F a x . + 497071293361, Abhreviatzons. TK, thymidine kinase; PCNA, proliferating cellnuclear antigen; DRB, dichlorobenzimidazole riboside.

S phase, occurs [7]. When the pOz is reduced below 150 ppm, cell damage occurs and the reversibility is lost [3, 41. The reversible shutdown of scheduled initiation events is possible at various stages of the S phase, even before the activation of the very first replicons [6]. In the latter case, the cells seem to arrest very close to the G1/S border, ready to activate normal S-phase replication within a few minutes after reoxygenation [6]. Cells of various stages of the GI phase obviously proceed to this point under hypoxic conditions and synchronously initiate the replicons scheduled to replicate as the very first of the S phase. A prominent feature of this regulation is the short time span between reoxygenation (after several hours of hypoxia) and the emergence of the replicon initiation burst. Therefore, we supposed that the O,-dependent replication control acts directly on the replication apparatus of the Ehrlich ascites cells. It seems to differ distinctly from the on/off switching of replication which occurs when cells alter from a resting (Go) state to the proliferative or cycling state where, after appropriate stimulation of resting cells, several hours elapse before DNA replication starts. During this time, a cascade of gene expression changes prepares cells for DNA synthesis [8]. However, it has not been examined so far whether alterations of gene expression are involved in the 02-dependent replication regulation. The main goal ofthe present work was to elucidate the role of gene expression in the oxygen-dependent regulation of replication in Ehrlich ascites cells. These studies were performed at two different levels. (a) We examined the level

390 of growth-regulated mRNA in the course of transient hypoxia experiments and compared the results with data reported in the literature for transitions from Go to S and from G I to S. Clearly, this could only be performed on selected examples. We choose three late mRNA species [S] coding for thymidine kinase (TK), a typical representant of growth-regulated enzymes providing building blocks for DNA synthesis and for proliferating cell-nuclear antigen (PCNA)/cyclin, a growthregulated component of the cellular replication machincry itself and for histone H3.1, a protein serving to package DNA after its replication. (b) By applying inhibitory drugs [DRB (dichlorobenzimidazole riboside), cycloheximide and puromycin] we examined the dependence of replicon initiation on ongoing transcription and translation in normal aerated cells as well as in cells subjected to hypoxia and reoxygenation, respectively, by two independent methods; analysis of the length distributions of growing daughter-strand DNA by alkaline sedimentation and DNA-fibre autoradiography. Neither hypoxia nor reoxygenation induced relevant changes in the expression of TK and PCNA/cyclin mRNA, respectively. Histone H3.1 expression seemed to follow the alterations of DNA replication imposed by transient hypoxia protocols. Replicon initiation, but not chain growth in active replicons and DNA maturation of Ehrlich ascites cells, depends on a very short-lived product of ribosomal protein synthesis which is also formed under hypoxic conditions. The RNA message for the latter seems to be relatively long-lived. Apparently, the condition in resting hypoxic Ehrlich ascites cells represents a particular sub-state of the S phase, differing from the latter only by a suppression of replicon initiations.

MATERIALS AND METHODS The following matcrials and procedures were described earlier: Ehrlich ascites cells and the selection of synchronous subpopulations by zonal centrifugation [9]; culturing of asynchronous and synchronous cells under ‘controlled hypoxia’ and reoxygenation, radioactive labeling and counting procedures, DNA flow cytometry [ 3 ] ;sedimentation analysis of radiolabeled daughter DNA chains after cell lysis on alkaline sucrose gradients [I]; DNA fibre autoradiography and evaluation of the autoradiographic patterns [2, 101. 2-3 h before experiments, asynchronous cells were routinely resuspended at 4 - 6 x 105/ml in 75% fresh/25% old medium. Thc term ‘controlled hypoxia’ means the O2 conditions occurring in a cell culture gassed with an artificial atmosphere containing 200 ppm O2 only. A detailed analysis of the behaviour of the pOz in such gassed cultures is given in [3].Cycloheximide (Boehringer, Mannheim) and puromycin (Sigma) were added to normal aerated cultures in the form of freshly prepared aqueous solutions (300-fold concentrated). The solvent used for DRB (Sigma) was ethanol. For hypoxic cultures, the dissolved drugs were evenly spread on small glass plates (25-mm diameter) and dried. Before establishing hypoxia, these plates were mounted above the culture fluid and, at the desired time, immersed without opening the culture vessels. RNA was extracted from cell samples according to [lI ] and separated, according to [12], into poly(A)-rich and poly(A)depleted fractions by chromatography on small oligo(dT) cellulose columns containing about 50 mg (dry mass) adsorbent (Pharmacia, Type 7), precipitated with 2.5 vol. ethanol, redissolved in 0.1 mM EDTA and quantified by measuring the A260nm. All RNA samples from an experiment were

electrophoresed in parallel lanes of 1 % agarose/b% forinaldehyde gels (2 pg poly(A)-rich or 4 pg poly(A)-depleted RNA/ lane), electroblotted to Hybond N + membranes (Amersham), fixed by ultraviolet radiation using a Stratalinker (Stratagene) as recommended by the supplier and thereafter additionally ‘baked’ at 80°C for 1 h. These membranes could be reprobed, after boiling in 0.1% SDS, up to six timer without significant loss of signal intensities Blots carrying poly(A)-rich RNA for estimating the mRNA of ‘TK or PCNA/cyclin were first probed with a 8actin probe. If the intensities of the p-actin signals exhibited distinct differences, electrophoresis was repeated with accordingly adjusted amounts of RNA, providing nearly identical signal strengths. No adjustment was necessary in the case of poly(A)-depleted RNA blots serving for the estimation of histone H3.1 mRNA. The probes used were purified plasmid inserts or parts of inserts: TK, 1.2-kb mouse cDNA clone muTK [13], excised by EcoRI from the vector pUC18; PCNA/ cyclin, human, 1.3-kb cDNA clone S14 [I41excised by BumHI from the vector pUC19; histone H3.1, human, genomic, a 0.4-kb portion containing most of the coding region was excised by StuI from the LK288 clone [I51 in pBR322; 8-actin, 2.1-kb human cDNA excised by BamHI from the clone pHFfiA1 [16] in the vector pCD. Random oligonucleotide-primed radiolabeling of the probes by [w3’P]dCTP (Amersham) to a specific activity of 1 - 2 x lo9 dpmlpg and hybridisation were performed as described [Ill. The sizes of‘ the mRNA species detected on the blots were estimated by using a 0.24-9.5-kb RNA ladder (Gibco-BRL) as reference. The suitability of pactin hybridisation of poly(A)-rich RNA blots for the control of gel loading was tested by comparing the signal strengths obtained by analyzing strictly identical amounts of total RNA yielded from experiments using different methods. Both methods did not alter the p-actin signals significantly.

RESULTS Expression of mRNA of cell-growth-related genes in transient hypoxia experiments We used two different protocols for treatment of cells before RNA extraction for Northern-blot analysis. Protocol I This started with a large cell culture, 2 h after renewal of 75% of the medium. 15% of the culture was separated a s an euoxic control (sample F).The rest was subjected to controlled hypoxia. Hypoxic cell samples were drawn after 3.5 h and 12 h (samples A and B). The remaining cells were reoxygenated at 12 h and samples were drawn from them 15, 40 and 120 min after reoxygenation (samples C - E). Protocol 2 This started with GI cells selected by a zonal-centrifugation procedure described earlier [9, 171. The selected cells were suspended in preconditioned medium, subjected to 12 h controlled hypoxia. then reoxygenated. Samplcs were drawn from the hypoxic cells after 12 h hypoxia (sample S,, ‘pre-S cells’) and from the reoxygenated cells 30 min and 120 min after restoring the atmospheric O2 tension (samples S, and Sb). Detailed descriptions of the effects of these treatments on replication of Ehrlich ascites cells have been published [2- 4,

391

Fig. 1. Influence of transient hypoxia on mRNA levels of some growthregulated mRNA species. Protocol 1 (for details o r the transient hypoxia protocols see Materials and Methods), (A) 3.5 h and (B) 12 h hypoxia; (C) 15 min, (D) 40 min and (E) 2 h after reoxygenation following 12 h hypoxia; (F) untreated control. Protocol 2, (S,) G1 cells after 12 h hypoxia, same cells 30 min (S,) and (S,) 2 h after rcoxygenation. contr.. control analyses of G1 cells (GI) and S cells (S) without hypoxic treatment. The blot regions shown correspond to the gel positions to which RNA of the indicated chain length (reference: RNA ladder 0.24-9.5 kb) migrated. Blots from gels run with poly(A+) RNA were first probed with the 8-actin reference probe and, after boiling in 0.1 % SDS, with the probe indicated. Histone H3.1 (Histon H3.1) was probed on blots from gels run with poly(A-) KNA without using a reference.

6,7,18]. It was ensured by appropriate control analyses (DNA flow cytometry, alkaline-sedimentation analysis of growing daughter strand DNA, kinetics of the [3H]dThdincorporation rate) that these effects also occurred in the present experiments. Protocol 1, after 12 h hypoxia, produced a mixture of cells accumulated at the G1/S boundarp (‘pre-S’ cells) and of cells arrested in different stages of the S phase (‘So’cells). The DNA synthesis rate of such a cell culture is usually decreased to much less than 5% that of the aerated control cells. After only 3.5 h hypoxia, this condition is not yet attained (still about 50% DNA synthesis but already severe suppression of replicon initiations). The burst of replicon initiations occurring after reoxygenation following 12 h hypoxia usually elevates the DNA synthesis rate within 2 h to above 300% of the control, because, at the same time, So cells resume, and accumulated pre-S cells synchronously begin S-phase replication. The zonal-centrifugation procedure providing the starting cells for protocol 2 usually yields G I cells which are greater than 95% pure [3].At the end of the following 12 h hypoxic period, these cells still exhibit the same GI DNA content as directly after selection and do not detectably incorporate [3H]dThd [3, 61. Reoxygenation triggers synchronous replicon initiations in this population of ‘pre-S’ cells and releases them into a normal S phase followed by at least two further synchronous cell cycles [6]. We performed two independent protocol-1 experiments, two independent protocol-2 experiments and one separate control experiment examining the mRNA in question in GI and S cells, repectively, as thcy had been selected by zonal centrifugation (samples G I and S). The amounts of RNA isolated in these experiments were sufficient for several gel runs (and blots). Fig. 1 shows examples of the Northern-blot signals obtained. All evaluated blots unambiguously indicated that reoxygenation after 12 h hypoxia causes no increase of the steadystate level of mRNA coding for TK and PCNA/cyclin. The level of the PCNA/cyclin mRNA never changed significantly,

neither under protocol 1 nor under protocol 2. However, the Northern-blot signals obtained with the TK probe indicated that this mRNA slightly increased over 3.5 - 12 h hypoxia in the course of protocol I when increasing amounts of ‘pre-S’ cells accumulated, and slightly decreased over 30 - 120 min after reoxygenation in the course of protocol 2, when ‘pre-S’ cells synchronously traversed the S phase. During the first mentioned hypoxic period, the GI compartment of the asynchronous population moves close to the G1/S border to accumulate in the ‘pre-S’ state, and during the last mentioned reoxygenated period released ‘pre-S’ cells proceed highly synchronously through the S phase [3, 61. Fig. 1 shows the signal serics most distinctly exhibiting these changes. The blots from the remainder of the independent experiments indicated less pronounced differences in each case. The control experiment with untreated G I and S cells revealed a significantly elevated level of TK mRNA in the early S phase in relation to G1. An analogous, but less pronounced, difference was found with the PCNA/cyclin probe. The mRNA level for histone H3.1 was also higher in S cells than in G1 cells. In contrast to the mRNA of TK and PCNA/cyclin, this mRNA distinctly decreased in the protocol-1 experiments under hypoxia and reincreased after reoxygenation. Note that not until 2 h after reoxygenation, when the DNA synthesis rate already exceeded that of the control by a factor of 3, did the H3.1 mRNA level attain that of the control. The same delayed increase was found in the protocol2 experiments. Thus, the increase in the H3.1 mRNA appears to succeed rather than to precede the rapid increase in DNA synthesis triggered by the reoxygenation. Replication after inhibiting transcription or translation

In preliminary experiments, the Ehrlich ascites cells used proved to be largely resistant to a-amanitin, perhaps by defective uptake of the drug. Therefore, we exclusively used 100 pM DRB to inhibit transcription. All experiments imposing a block of translation were performed in parallel with 30 pM cycloheximide and 100 pM puromycin. Both drugs yielded virtually the same results. In the following, we present the results obtained with DRB and cycloheximide. Fig. 2 shows the kinetics of incorporation of radioactivity into acid-insoluble material from [3H]Urd and [3H]dThd, respectively, after DRB addition and the kinetics of [ 3H]dThd incorporation after addition of cycloheximide. DRB caused a fast decline in [3H]Urd incorporation. Although 100 pm DRB also causes about 30% inhibition of Urd uptake into animal cells [20], the [‘HIUrd incorporation curve indicates, according to [20], a practically complete arrest of m R N h formation within a few minutes of drug addition. Since RNA polymerase I1 is distinctly more sensitive to DRB than the other cellular KNA polymerases [19,20], the remaining [3H]Urd incorporation concerns essentially non-mRNA species. Nevertheless, the DRB effect on DNA replication emerged relatively slowly over the course of several hours. In contrast, cycloheximide exerted the most marked depression of DNA synthesis already within the first hour. However, the decline still continued during the rest of the observed period, in a manner that would be expected if only replicon initiation is suppressed, and chain elongation continues normally [21]. Data presented below in Table 1 and results published earlier [6] demonstrate that exactly this occurs in cycloheximide-treated Ehrlich ascites cells. In order to resolve the first minutes after cycloheximide addition, we used a continuous radiolabeling schedule which is more suited to analyze fast changes in the incorporation

392

1

2

3 time

4

5

6

(h)

Fig. 2. I3HldThd incorporation into acid insoluble material by aerated cells after addition of DRB and cycloheximide and I3HIUrd incorporation after DRB addition. At the times indicated after drug administration, 1 ml aliquots of parallel cultures (drug treated and control) were labeled for 10 min with 10 pCi [3H]dThd 2 pM dThd and processed for determination of acid-insoluble 'H. Incubation of samples at time zero was started immediately before drug addition. The incubation with [3H]Urd (5-'H, 30 pCi, 20 Ci/mmol) was performed in a separate experiment. ( 0 )100 pm DRB, [3H]dThd;(a)30 pm cycloheximide, 100 iim URB, l3H]LTrd. [jH]dThd; (0)

+

rate of radioactive labels. Fig. 3 shows that cycloheximide caused an almost immediate complete inhibition of protein sythesis (represented by the [3H]Leu incorporation) whereas DNA labeling with [3H]dThd was not significantly suppressed during the first 5 min before it decreased to about 20% of the control rate. In contrast to hypoxia, cycloheximide does not influence dNTP pools of Ehrlich ascites cells (Probst, H., Brischwein, K. and Engelcke, M., unpublished results), and therefore [3H]dThd incorporation directly reflects DNA synthesis. 'Thus, the decrease in DNA synthesis caused by cycloheximide is delayed by a short but significant interval of about 5 rnin as compared to the immediate complete inkbition of protein synthesis. It is worth mentioning that the course of the curve shown in Fig. 3B could not be altered at all by pretreatment of the cells with high doses ofthe inhibitors of intraceilular proteolysis leupeptin, antipain and pepstatin [22], although these inhibitors, in control experiments, significantly prolonged the lifetime of [3H]Leu-labeled protein in Ehrlich ascites cells (data not shown). Fig. 4 shows the effect of DRB administration on the length distribution of growing daughter DNA chains, labeled by an 8-min pulse with [3H]dThd and analyzed by alkaline sedimentation. No significant changes occurred during the initial 90 min. Only beyond 2 h after administration, did DRB progressively diminish the relative proportion of the slowly

Table 1. Results of DNA-fibre autoradiography. The cells were labeled with [3H]dThd according to a 20/20 rnin hot/warm prolocol [2, 10, 401. Thc paramcter called relative initiation frequency is the ratio of post-pulse figures to pre-pulse figures.

Treatment of cells

Relative initiation frequency

10 h normal aerated growth, euoxic labeling, (euoxic control)

1.21

10 h hypoxia, hypoxic labeling, (hypoxic control)

0.09

10 h hypoxia, 1 h rcoxygenated growth, euoxic labeling

2.70

9 h hypoxia, 1 h 150 pM DRB t hypoxia, I h reoxygenated growth, euoxic labeling

0.50

6 h hypoxia, 4 h 150 pM DRB + hypoxia, 1 h reoxygenated growth, cuoxic labcling

0.23

9 h 50 min hypoxia, 10 min 30 pM cyclohexirnide + hypoxia, 1 h rcoxygenated groat h , cuoxic labcling

0.10

9 h 30 rnin hypoxia, 30 min 30 pM cycloheximide + hypoxia, 1 h reoxygenated growth, euoxic labeling

0.03

n

Fork progress rate

159

pm/min 0.84 (mean) 0.22 (SD)

131

0.69 (mean) 0.29 (SD)

n

152

290

Inter-initiation distance Pm 82.2 (mean) 39.7 (SD) 78.7 (median)

n

48

100.8 (mean) 53.2 (SD) 80.2 (median) 53

674

365

61.8 (mean) 41.7 (SD) 49.5 (median)

444

8 1.9 (mean) 46.7 (SD) 69.8 (median)

0.75 (mean) 0.35 (SD)

388

333

0.78 (mean) 0.33 (SD)

207

147

0.88 (mean) 0.23 (SD) 234

98.3 (mean) 36.2 (SD) 95.4 (median)

80

153

0.65 (mean) 0.19 (SD)

278

94.2 (mean) 63.3 (SD) 83.2 (median) 90

133

0.74 (mean) 0.20 (SD) 258

122.6 (mean) 72.0 (SD) 110.3 (median) 76

393 f

5

A 4 U

P

\

m

I

3

0

1

32

1

B y

24

4

\

m 0

n

16

u

Y

/

0

2

8

1

I

5

10

15

time

20

25

30

35

Imlnl

5

10

15

time

20

25

30

35

(minl

Fig.3. Effect of cycloheximide on the incorporation of I3HJLeu (A) and I3WdThd (B) into acid-insoluble material in a continuous labeling experiment. A cell culture was labeled overnight with 1.5 nCi/ml [I4C]dThd and divided into four portions 3 h after furnishing them with 75% of fresh medium. At 0 min, 5 pCi/ml [3H]Leuwere added to two of the portions, and 10 pCi/ml [3H]dThd + 2 pM dThd to the remaining two samples. After 10 min, 30 pM cycloheximide were added to one of the [3H]Leu-labeled or the [3H]dThd-laheled portions, repectively. 1 ml samples were stopped at the times indicated and processed for determination of acid-insoluble I4C and 3H. ( 0 )Samples from untreated portions; (H) samples from portions treated with cycloheximide; broken line, addition of cycloheximide.

sedimenting short chains in the sedimentation profiles. Since, as shown in Table 1, DRB does not alter the fork progress rate of Ehrlich ascites cells, this indicates that replicon initiations which supply new short chains into the total population of growing chains became progressively scarce beyond 2 h after DRB addition to the cells. Figs 4D, H, I show in addition that DRB does not influence DNA maturation. The sedimentation profiles H and I (Fig. 4) exhibit normal maturation [18, 231 and do not significantly differ from each other and from the untreated control (Fig. 4G). Analogous experiments using cycloheximide and puromycin (puromycin data are not shown) demonstrated (Fig. 5 ) that a block of translation significantly diminished the short chains originating from newly initiated replicons as soon as 15 min after administration of the drug. As chain growth in active replicons must occur for several minutes to reveal a decreased number of initiations in the sedimentation profiles of growing chains, cycloheximide appears to suppress replicon initiation much earlier than 15 rnin after drug administration. Later, the peak of the profiles shifted progressively towards higher S values and did not attain a plateau pattern as in the case of DRB treatment (Fig. 4). Since the rate of fork progression is not affected ([6] and Table l),this indicates that cycloheximide causes an abrupt and complete suppression of replicon initiations. In the case of DRB treatment, however, the progressivc flattening of the profiles indicates that suppression of replicon initiations occurred rather gradually. It has already been published [6] and reproduced again in the present context, that translation inhibition lasting for several hours also has no influence on DNA maturation in Ehrlich ascites cells (data not shown). Furthermore, the inhibitors of intracellular proteolysis mentioned above did not change the profiles in experiments analogous to that of Fig. 5 (not shown).

Combination of transcription or translation blocks with transient hypoxia Alkaline sedimentation profiles

As demonstrated earlier [1- 3, 61, hypoxia lasting for several hours greatly diminishes the number of active replicons

in Ehrlich ascites cells. The chain-length distributions of daughter chains in the few replicons still found active are accentuated in the region of long molecules originating from rather ‘old’ replicons. This state is represented by profile A obtained in the experiment of Fig. 6. (The euoxic control is not shown; the profile obtained was virtually identical to that shown in Figs 4A and 5A.) Reoxygenation reproducibly triggers the initiation of a large number of new replicons which incorporate, 40 min later, usually greater than 2/3 of the total radioactivity appearing as a new peak around 40 S (Fig. 6B). Addition of DRB before O2 recovery reduced the proportion of new ‘young’ replicons in a manner dependent on the time between DRB addition and reoxygenation, but, in accordance with the results of the experiment of Fig. 4, did not abolish them, even after 4 h treatment (Fig. 6 C and D). In contrast, only 5 min treatment with cycloheximide was distinctly more effective than 4 h treatment with DRB. A very small proportion of short chains from young replicons is just visible in the profile shown in Fig. 6 E. This small proportion could be visualized better than in Fig. 6E in another experiment also employing 5 min of cycloheximide treatment before reoxygenation, but allowing only 20 min reoxygenated growth before radioactive labeling (profile not shown) instead of 40 min in the experiment of Fig. 6. Addition of cycloheximide 30 min before reoxygenation, however, essentially completely abolished the ability of the cells to activate new replicons upon reoxygenation. Intervening replicon terminations greatly diminished the total replicative activity found in such cells 40 rnin after reoxygenation (i. e. 70 rnin after cycloheximide addition). Fig. 6 F shows that the residual chain growth occurred essentially only in ‘old’ replicons. (The slowly sedimenting radioactivity appearing in the very first gradient fractions of profile 6F represents Okazaki fragments [24].) In the experiment of Fig. 7 , we examined the effect of cycloheximide when it was added subsequently and 30 min after reoxygenation. The pulse-labeling window covered the period directly after cycloheximide addition and 40 - 50 min thereaftcr. Addition of cycloheximide concurrently with reoxygenation still allowed a (diminished) initiation burst to occur (Fig. 7 C ) . In Fig. 7 C , the proportion of radioactivity in the flank of the lowest S values was diminished preferably

394

A

-

E R

a

I

0

F

0

s

10

is

20

25

30 5 i n 15 f r a c t i o n number

20

25

30

5

10

15

20

25

30

(from top)

Fig. 4. Alkaline-sedimentation analysis of the lengths of growing daughter strands, effect of DRB. Part of a cell culture was incubated with 100 pM DRB, the rest served as a control. At the limes indicated below, 1 ml samples were pulse labclcd for 8 rnin (samples A - F) or pulse/ chase labeled (8 min/90 min, samples G- I) with 30 pCi [3H]dThd and the cells were lysed on the top of alkaline-sucrose gradients for sedimentation analysis. In the profiles depicted, the sedimentation direction i s from left to right. A 39.5 S sedimentation marker (14C phage i, DNA) was reproducibly recovered in fraction 9. (A) Untreated control, pulse labeled after 5 h; (B-F) pulse labeling after 45 iiiin (B), 2.5 h (C), 2 h (D), 4 h (E) and 8 h (F) incubation with DRB; (G) untreated control, pulse/chase labeled after 4 h; (H, I) pulse/chase labeling after 3 h (H) and 5 h (I) incubation with DKB. Total radioactivity recovered from the gradients: A, 52010; li, 44361; C, 60472; D, 20268; E. 13040; F, 3922; G, 29477; H, 20240; I, 18836cpm.

as compared to the control (Fig. 7A). This indicates the bcginning of resuppression of replicon initiations within the 8 min pulse. SO rnin after (Fig. 7D), the main peak was shifted to about 70 S and the total recovered radioactivity was diminished to less than 30% of the initial state (Fig. 7C) and less than 10% of the corresponding control profile (Fig. 7B). Thus, preferably few ‘old’ replicons were still active under thcsc conditions. Also, when cycloheximide was added 30 min after reoxygenation (Fig. 7 E and F) the sedimentation profiles indicate that the resuppression of replicon initiations had already begun earlier than 8 min after the cycloheximide addition. It is reasonable that the long labeled chains dominating the Fig. 7 D and F originate, for the most part, from replicons initiated after O2 recovery and before the onset of the cycloheximide effect. Afterwards, they obviously grew to their respective size as a more or less synchronous wave. In summary, the experiments combining transient hypoxia and cycloheximide treatment demonstrate that translation must be blocked more than 5 min before reoxygenation in

order to prevent the initiation burst which normally follows O2 recovery. This implies that the formation of a bhort-lived product of ribosomal protein synthesis which, obviously, is necessary for replicon initiation, is not affected by the hypoxia. DNA$bre autoradiography Table 1 shows the results of the statistical evaluation of the autoradiographic patterns obtained in an experiment designed in analogy to that of Fig. 6 . The data providc compelling evidence that hypoxia, neither alone nor in combination with DRB or cycloheximide treatment, has any significant effect on the progression rate of replication forks in the F,hrlich ascites cells. In contrast, the relative frequency of rcplicon initiations as reflected by the ratio of ‘postpulse’ to ‘prepulse’ initiation patterns [2, 251 differs by almost two orders of magnitude. In accordance with results reported earlier [2], 10 h hypoxia reduced this ratio in relation to the euoxic control by more than an order of magnitude. Reoxygenation re-increased

395 10 -

8'

B

5 1 1 0 -

10

15

20

25

30

10

L, rl

>

n

u o

-

0

8-

8

c

6-

D

6

U

-5

10

I5

20

25

30

f r a c t i o n number

--5 10 15 20 [from top)

25

30

Fig. 5. Alkaline-sedimentation analysis of lengths of growing daughter strands, effect of cycloheximide. 8 rnin pulse labeling with 30 pCi/ml ['HIdThd after incubation with 30 pM cycloheximide Tor 15 rnin (B), 45 rnin (C) and 90 min (D); A, untreated control. Total radioactivity recovered from the gradients: A, 67781; B, 25618; C, 20027; D, 14362 cpm. For further details see legend of Fig. 4.

this value to more than double the value of the control. This re-increase was suppressed to varying extents by addition of DRB or cycloheximide and was dependent on the time interval between drug administration and reoxygenation. Again, 4 h treatment with DRB was not as effective as a 10-niin treatment with cycloheximide, which completely prevented the relative frequency of initiation being attained when the cells were reoxygcnated. 1 h treatment with DRB had a slight but significant effect. 30 rnin treatment with cycloheximide caused an cxtra suppression of replicon initiation, exceeding that of hypoxia alone. The inter-initiation distances exhibited a clear tendency to increase when the relative initiation frequency decreased and vice versa.

DlSCUSSION Expression of mRNA of growth regulated genes The genes whose cognate mRNA are induced when quiescent cells are stimulated to proliferate can be conveniently divided into two large groups (for a review see [S]): the early growth-regulated genes, to which belong, for instance, c-fos and c-myc, and the late growth-regulated genes coding for the most part for proteins directly or indirectly involved in DNA replication, for instance enzymes of the nucleotide metabolism, diverse polymerases and other cofactors of the cellular replication machinery. The mRNA for a number of histones are frequently also assigned to the late group. However, the levels of some histone mRNA seem to also depend on ongoing DNA replication [26] justifying to assign these messages to a separate group called 'replication-dependent' mRNA. The mRNA coding for TK is perhaps the most intensively studied example of a late-growth-regulated mRNA [S, 27 -

F

.

.

.

.

.

.

.

.

.

,

5

10

15

20

25

30

5

LO

15

20

fractlon number

, 25

, 30

(from t o p )

Fig. 6. Alkaline-sedimentation analysis of lengths of growing daughter strands, effect of addition of 100 pM DRB (C, D) and 30 pM cycloheximide (E, F) on the restoration of replication upon reoxygenation after 10 h hypoxia. Pulse labeling for 8 min with 30 pCi/ml [3H]dThd. A (control), labeling before reoxygenation; B (control), labeling 40 rnin after rcoxygenation; C, as B, DRB addition 1 h before reoxygenation; D, as B, DRB addition 4 h before reoxygenation; E, as B, cycloheximide addition 5 min before reoxygenation; F, as B, cycloheximide addition 30 rnin before reoxygenation. Total radioactivity recovered from the gradients: A, 16452; B, 46472; C, 27916; D, 12609; E, 14258; F, 3492 cpm. For further details see legend of Fig. 4.

331. The expression of TK mRNA is frequently taken as being indicative of the growth state of mammalian cells [XI. As far as is known, the mRNA coding for PCNA/cyclin behaves very similarily [8,14,34 - 361. These two mRNA species are usually easily detectable in continuously cycling cells, frequently exhibiting lower levels in the GI state than in the S phase. The latter was confirmed in our control analyses of untreated G and S cells (Fig. 1). In quiescent cells, these inRNA species often are barely detectable. After induction of quiescent cells to re-enter the cycle, a steep increase shortly before the onset of DNA synthesis marks the end of the chain of events [Z] leading from Go to the beginning of a new S phase. In our experiments (Fig. l), no changes of the levels of TK or of PCNA/cyclin mRNA which are characteristic of a G1- S or a Go-S transition [S], respectively,were induced by reoxygen-

396 assumption that regulation of this message is dependent on the ongoing DNA replication [26].

10

8

B

Influence of inhibitors of transcription and translation on replicon action in aerated and transiently hypoxic cells

6

4

-

2

E

0.

D

E

F

L ,

5

LO

15

20

25

30

f r a c t l o n number

5

10

15

20

.

25

.

30

(from top)

Fig. 7. Alkaline-sedimentation analysis of lengths of growing daughter strands and the effect of addition of 30 pM cycloheximide to reoxygenated cells after 10 h hypoxia concurrently with readmission of Oz (C, D) and 30 min thereafter (E, F). A (control, no cycloheximide addition), coincidcnt start of reoxygenation and pulse labeling (30 pCi/ml [3H]dThd, 8 min); B (control, no cycloheximide addition), pulse labeling 50 min after reoxygenation: C, D. as A. B but cycloheximide addition together with reoxygenation : E, labeling start and cycloheximide addition 30 min after reoxygenation; F, cycloheximide addition 30 min after reoxygenation, labeling 70 min after reoxygenation. Total radioactivity recovered from the gradients: A, 23412; B, 51 103; C, 16332; D, 451 1 ; E, 30 859; F, 1 1 638 cpm. For rurther details see legend of Fig. 4.

ation of hypoxic cells. Although about 50% of the asynchronous cells subjected to protocol 1 and nearly all cells subjected to protocol 2 are in the G I compartment at the beginning of hypoxic treatment [3], absolutely no increase in expression preceded or accompanied the burst of initiations triggered by reoxygenation in hypoxic cells treated according to either protocol. These G I cells obviously progressed normally to (enhanced) S-phase-like expression of the examined lategrowth-regulated mRNA, despite the hypoxia. When they were reoxygenated after 10 - 12 h hypoxia, the expression state attained obviously was not altered to allow for the subsequent start of DNA synthesis. The subsequent delay of histone H3.1 mRNA expression to changes in the replicative activity imposed by changes in the pOz is compatible with the

It has been known for almost two decades that mammalian DNA replication depends on ongoing protein synthesis [21, 37 -441. Some studies also examined the dependence of mammalian DNA replication on RNA synthesis [45 -471. The first data on Ehrlich ascites cells was published by us [6] indicating that cycloheximide treatment, in close analogy to hypoxia, specifically suppresses replicon initiation in these cells. The present study is intended to elucidate this peculiar coincidence. On the basis of the kinetic curves, the alkaline sedimentation profiles, the data from DNA-fibre autoradiography presented in Figs 2 - 7 and in Table 1 and taking into account the data published by us earlier [2, 3, 61 we can state the following. (a) The replication machinery of Ehrlich ascites cells responds to controlled hypoxia as well as to inhibition of transcription or translation by an isolated suppression of replicon initiation. DNA-chain growth and maturation still continue, at least for several hours, essentially undisturbed. (b) A relatively small, but clearly defined delay of about 5 min separates the onset of an essentially complete inhibition of protein synthesis from the beginning of a rapid failure of replicon initiations. The suppression of replicon initiations caused by inhibition of transcription, in contrast, does not become detectable until 1-2 h after drug administration, and henceforth increases. The lag between the relevant changes in the pOz and the on/off switching of replicon initiation is also rather short, in the range of a few minutes [ 2- 41. (c) Inhibition of transcription or translation in hypoxic cells, prevents the burst of replicon initiations normally occurring upon rcoxygenation. However, the transcription inhibitor must be added several hours before reoxygenation, while addition of a Lranslation inhibitor can be made as close as about 5 min before O2 readmission. (d) The initiation burst triggered by reoxygenation can be re-interrupted by inhibiting translation shortly after oxygen recovery. The re-interruption, producing a single surge of daughter-chain growth, becomes effective within a few minutes after drug administration. A similarly fast reinterruption cannot be effected in inhibiting transcription (data not shown). On the basis of these statements, we conclude that replicon initiation in Ehrlich ascites cells depends (a) on a minimal O2 tension and (b) on the availability of at least one product of ribosomal protein synthesis which exhibits, at least in the active form, a very short lifetime in the range ofa few minutes. The RNA message(s) for the protein(s) concerned is (are) rather long-lived with lifetime(s) in the range of several hours. The availability of this mRNA, as well as that of the shortlived protein(s) is not affected by several hours of hypoxia. It remains open whether the short-lived protein factor itself is an essential component of the initiation apparatus or whether it functions by activating one or several components of them, for instance by modification. It is also unclear which mechanisms cause its short lifetime. The ineffectiveness of leupeptin, antipain and pepstatin does not exclude proteolytic degradation since these substances inhibit only a part of the known intracellular proteolytic activities, a great number of which play important roles in cellular regulation [48]. However, the factor could also be inactivated by modification reactions or, if directly involved in the processes occurring at the replicon origins, be sequestered by becoming durably

397 bound to specific DNA sites, as was described, for example, for the large T-antigen of Simian Virus 40 in the replication of a chimeric Simian-Virus-40 - bovine-papilloma-virus episoma1 replicon [49]. Amongst the cell lines examined so far, only Ehrlich ascites cells exhibit a sole dependence of replicon initiation on shortlived protein. In other cells, replicon initiation and the rate of propagation of replication forks, are both involved [21, 37441. In Simian-virus-40 replication in vivo, initiation and fork propagation were found not to be dependent on short-lived protein, whereas in host genome replication, replicon initiation was rapidly suppressed and fork propagation was concurrently slowed down by cycloheximide [50].Consequently, it was supposed that the large T antigen, having initiator functions and fork-propagation (helicase) functions as well, substitutes in viral replication for short-lived host protein(s) involved in the hosts own replicon initiation and fork propagation. It can be speculated that other (long-lived) cellular helicases possibly replace, in cellular replication, the shortlived fork-propagation function more or less sufficiently, whereas no substitute of the initiator function is available. This could explain why cycloheximide suppresses cellular replicon initiation in many cell types almost completely but only slows down, more or less completely, their fork propagation. The mechanism by which the O2 tension acts on replicon initiation is, to date, not definitely clarified. However, there are suggestions [I81 that the changes of p 0 2 alter ribonucleotide reductase activity in living cells and thereby induce changes of a (as yet unknown) deoxycytidine derivative which is suspected to have a positive effector function in replicon initiation. It is also unclear so far whether a short-lived protein is directly involved in the 02-dependent regulation of replication. However, recent DNA fibre autoradiographs of HeLa and RHK cells (not shown) indicate that hypoxia, in Ehrlich ascites cells only suppressing replicon initiation, also reduces fork propagation in these cells to the same extent as docs cycloheximide. In the light of the above mentioned dual function of the T antigen, this parallelism of hypoxia and the cycloheximide effects favors the possibility that a short-lived protein is involved in mediating the POz-dependent response of the replication machinery.

ment without requiring that the replicative or cycling state has to be left. For providing gene probes, we thank to Dres. L. Kedes (histone H3.1, p-actin), R. Bravo (PCNA/cyclin) and E. Wintersberger (TK). Leupeptin, antipain and pepstatin were kind gifts of Dr P. Bohley. This work was supported by the Deutsche Forschungsgemeinschuft (Pr 95/10-3).

REFERENCES

1. Probst, H. & Gekeler, V. (1980) Reversible inhibition of replicon initiation in Ehrlich ascites cells by anaerobiosis, Biochern. Biophys. Res. Commun. 94, 55 - 60. 2. Probst, H., Gekeler, V. & Helftenbein. E. (1984) Oxygen dependence of nuclear DNA replicalion in Ehrlich ascites cells, Exp. Cell. Res. 154, 327 - 341. 3. Probst, H., Schiffer, H., Gekeler, V., Kienzle-Pfeilsticker, T I . , Stropp, U., Stoker, K.-E. & Frenzel-Stotzer, I. (1988) Oxygen dependent regulation of DNA Synthesis and growth of Ehrlich ascites cells in vitro and in vivo, Cancer Res. 48, 2053 --2060. 4. Probst, 13. & Gekeler, V. (1988) Oxygen dependent regulation of DNA replicalion of Ehrlich ascites cells in vitro and in vivo, in Oxygen sensing in tissues (Acker, H., ed.) pp. 79 -92, SpringerVerlag, Berlin, Heidelberg. 5. Froese, G. (1962) The respiration of ascites tumor cells at low oxygen concentrations, Biochim. Biophys Acta 57,509 - 519. 6. Gekeler, V. & Probst, H. (1988) Synchronisation of replicons in Ehrlich ascites cells, Exp. Cell Res. 175, 97 - 108. 7. Probst, H., Riedinger, H.-J. & Gekeler, V. (1989) No significant overreplication occurs in Ehrlich ascites cells during and after reversal of hypoxia, Exp. Cell Res. 180, 563 - 568. 8. Pardee, A. B. (1989) GI events and regulation ofcell proliferation, Science 246, 603 - 608. 9. Probst, 13. & Maisenbacher, J. (1973) Use of zonal centrifugation for preparing synchronous cultures from Ehrlich ascitcs cells grown in vivo, Exp. Cell Res. 78, 335 - 344. 10. Probst, H., Blutters, R. & Fielilz, J. (1980) DNA replication in asynchronous and synchronous Ehrlich ascites cells in different conditions of growth, Exp. Cell Res. 130, 1- 13. 11. Gekcler, V., Frese, G., Diddens, H. & Probst, H. (1988) Expression of a P-glycoprotein gene is inducible in a multidrug resistant human leukemia ccll line, Biochem. Biophys. Res. Commun. 15.5, 754-760. 12. Aviv, H. & Leder, P. (1972) Purification of a biologically active globin messenger RNA by chromatography on oligothymidylic acid cellulose, Proc. Nutl Acud. Sci. IJSA 69, 1408- 1412. 13. Hofbauer, R.. Mullner, E., Seiscr, C. & Wintersberger, E. (1987) Final remarks Cell cycle regulated synthesis of stable mouse thymidine kinase mRNA is mediated by a sequence within the cDNA, Nucleic According to the present results, the expression state of Acids Res. I S , 741 -752. genes, conditioning cells for DNA replication, seeins to be ‘replicative’ in Ehrlich ascites cells subjected to several hours 14. Ahnendral, J. M., Huebsch, D., Blundell, P. A., MacDonaldBravo, H. & Bravo, R. (1987) Cloning and sequence of the of hypoxia. This concerns the growth-regulated mRNA as human nuclear protein cyclin: Homology with DNA binding well as the (so far unidentified) short-lived protein(s) essential proteins, Proc. Nutl Acud. Sci. U S A 84, 1557- 1579. for replicon initiation. Apparently, the cellular replication 15. Wells, D. 62 K.edes, L. H. (1985) Structure of a human histon machinery is, during hypoxia, completely equipped to start cDNA: Evidence that basically expressed histone genes have replicon initiation, but an additional event, crucial [8] for intervening sequcnces and cncode polyadenylated mRNAs, rcplicon initiation, cannot take place as long as the Oz tension Proc. Nut1 Acud. Sci. U S A 82, 2834-2838. is below a critical threshold whch lies, however, above the 16. Gunning, P., Ponte, P., Okayama, H., Engel, J., Blau, H. & Kedes, L. (1983) Isolation and characterization of full length pOz depressing mitochondria1 respiration and influencing the c D N h clones for human alpha-, beta-, and gamma actin adenylate energy charge of the cells [3]. Thus, the hypoxic mRNAs: Sceletal but not cytoplasmic actins have an aminoarrest of replication in Ehrlich ascites cells differs from the terminal cysteine that is subsequently removed, Mol. Cell. Bid. replication inhibition following more severe stress (e. g. heat 3,787 - 795. shock) or DNA damage. In organisms having a blood circu17. Prohst, H. & Maisenhacher, J. (1975) Selection of synchronous lation, the pOz seems to be a suited indicator of the general cell populations from Ehrlich ascites cells by zonal centrifugasupply situation of tissues. A lowered pOz may often be indication, in Methods in cell biology (Prescott, D. M.. ed.) pp. 173 tive of impending suboptimal growth conditions. The 02184, Academic Press, New York, San Francisco, London. dependent regulation of replication provides a possibility for 18. Probst, H., Schiffer, H., Gekeler, V. & Scheffler, K. (1989) Oxygen fast adaptation of cell growth to changes of the micro-environdependent regulation of mammalian ribonucleotide reductase

398

19.

in vivo and possible significance for replicon initiation, Biochem. Biophys. Res. Comrnun. 163, 334-340. Chodos, L. A,, Fire, A,, Samuels, M. & Sharp, P. A. (1989) 5,6dichloro-I -8-n-ribofuranosylbenzimidazole inhibits transcription elongation by RNA polymerase I1 in vitro, J . Bid. Chcm. 264,2250 2257. Tamm, I. & Shegal, P. B. (1978) Halobenzimidazole ribosides and RNA synthesis of cells and viruses, Adv. Virus Rex 22, 187258. Seale, R. L. & Simpson, R. T. (1975) Effects ofcycloheximidc on chromatin biosynthesis, J . Mol. Biol. 94,479 - 501. Umezawa, H. (1982) Low molecular-weight enzyme inhibitors of microbial origin, Annu. REV.Microbiol. 36, 75 -99. Kowalski, J. & Cheevers. W. P. (1976) Synthesis of heigh molecular weight DNA strands during S phase, J . Mol. Biol. 104, 603 - 615. Gckeler, V., Stropp, U. & Probst, H. (1986) Application of hypoxia-induced shut down o f replicon initiation to the analysis of replication intermediates in Ehrlich ascites cells, Biol. Chem. Hoppe-Seyler 367, 1209- 1217. Richter, A. & Hand, R. (1979) DNA rcplication during a seruminduced S phase in primate CV-1 cells, Exp. Cell Res. 121, 363 -371. Osley, M. A. (1991) The regulation of histone synthesis in the cell cycle, Annu. Rev. Biochem. 60, 827 - 861. Coppock, D. L. & Pardee, A. B. (1985) Regulation of thymidine Kinase activity in the cell cycle by a labile protein, J . Cell. Physiol. 124, 269 - 274. Coppoek, D. L. & Pardee, A. B. (1987) Control of thymidine kinase mRNA during the cell cycle, M o l . Cell. Bid. 7; 29252932. Knight, G. B., Gudas, J. M. & Pardee, A. B. (1987) Cell-cyclespecific interaction of nuclear DNA-binding proteins with a CCAAT sequence from the human thymidine kinase gene, Proc. Nut1 Acad. Sci. USA 84,8350-8354. Stewart, C. J., Ito, M. & Conrad, S. E. (1987) Evidence for transcriptional and post-transcriptional control of the cellular thymidine kinase gene, Mol. Cell. Biol. 7, 1156- 1163. Kim, Y. K., Wells, S., Lau, Y.-F. C. &Lee, A. S. (1988) Sequences contained within the promoter of the human thymidine kinase gene can direct cell-cycle regulation of heterologous fusion genes. Proc. Nut1 Acad. Sri. USA 85, 5894- 5898. Sherley, J . S. & Kelly, T. J. (1988) Regulation ofhuman thymidine I Biol. . Chem. 263, 8350 8358. kinase during the cell cycle: . Dou, Q.-P., Fridovich-Keil, J. & Pardee, A. B. (1991) lnducible proteins binding to the murine thymidine kinase promoter in late G1/S phase, Proc. Natl Acad. Sci. USA 88, 1157-1161. Jaskulski, D., Gatti, C., Travali, S., Calabretta, B. & Baserga, R. (1988) Regulation of the proliferating cell nuclear antigen cyclin and thymidine kinase mRNA levels by growth factors, J. Biol. Chem. 263,1075 1079. ~

20. 21. 22. 23. 24.

25. 26. 27. 28. 29.

30. 31.

32.

~

33. 34.

~

35. Liu, Y.-C.. Marraccino, R. L., Kcng, P. C., Bambara. R. A., Lord, E. M., Chou, W.-C. & Zain, S. B. (1989) Requirement for proliferating cell nuclear antigen expression during stages of the Chinese hamster ovary cell cycle, Biochemistry 28,2967 2974. 36. Koniecki, J., Nugent, P., Kordowska, J. & Baserga, R. (1991) Effect of the SV40 T antigen on the posttranscriptional regulation of the proliferating cell nuclear antigen and DNA polymerase-rx genes, Cancer Res. 51, 1465-1471. 37. Weintraub, H. & Holtzer, H. (1972) Fine control of DNA synthesis in developing chicken red blood cells, J . M o l . Bid. 66, 13-35. 38. Hori, T.-A. & Lark, K. G. (1973) Effect of puromycin on DNA replication in Chinese hamstcr cells, J . Mol. Biol. 77, 391 -404. 39. Gautschi, J. R. & Kern, R. M. (1973) DNA replication in mammalian cells in the presence of cycloheximidc, Exp. Cell Res. 80,15-26. 40. Hand, R. & Tamm, I. (1973) DNA replication: Direction and rate of chain growth in mammalian cells, J . Cell. Bid. 5X, 410 418. 41. Gautschi, J. R. (1974) Effects of puromycin on DNA chain elongation in mammalian cells, J . Mol. Biol. 84, 223 - 229. 42. Hand, R. (1975) DNA replication in mammalian cells. Altered pattern of initiation during inhibition of protein synthesis, J . Cell Biol. 67, 761 -773. 43. Venkatesan, N. (1977) Mechanism of inkbition of DNA synthesis by cycloheximide in Balb/3T3 cells, Biochim. Biophys. Acta 478, 437-453. 44. Stimac, E. & Housman, D. (1977) Effects of inhibition of protein synthcsis on DNA replication in cultured mammalian cells, J . Mol. Biol. 115,485-511. 45. Seki, S. & Mueller, G. C . (1975) Requirement for RNA, protein and DNA synthesis in the establishment of DNA replicase activity in synchronized HeLa cells, Biochim. Biophys. Acta 378, 354- 362. 46. Guy, A. L. & Taylor, J. H. (1978) Actinomycin D inhibits initiation of DNA replication in mammalian cells, Proc. Null Acad. Sci. USA 75, 6088- 6092. 47. Hand, R . & Tamm, I. (1977) Inhibition of mammalian DNA replication by dichlorobenzimidazolc riboside, Exp. Cell Res. 107,343 - 354. 48. Bohlcy, P. (1987) Intracellular proteolysis. in Hydrol.viic enzymes (Neuberger, A. & Brocklehurst, K., eds) pp. 307-332, Elscvier Sciencc Publishers B.V. 49. Roberts, J. M. & Weintraub, H. (1988) Cis-acting negative control of DNA rcplication in eucaryotic cells, Cell 52, 397 - 404. 50. Schulte, D., Knippers, R., Dreier, T., Probst, G. & Probst, TI. (1992) Cycloheximide inhibits cellular, but no1 SV40, DNA replication, FEBS Lett. 299, 149- 154.