Hydrostatic pressure influences histone mRNA

ALISON L. SYMINGTON1, SELMA ZIMMERMAN2, JANET STEIN3, GARY STEIN3

and ARTHUR M. ZIMMERMAN1'*

^Department of Zoology, University of Toronto, Toronto, Canada MBS 1A12Dwiswn of Natural Sciences, York University, Ontario, Canada^Department of Cell Biology, University of Massachussets Medical Centre, Massachussets, USA

* Author for correspondence

Summary

Exposure of HeLa S3 cells to high hydrostaticpressure (6.89 xlO3 to 6.89xl04kPa: 1000 tolOOOOlbfin"2) reduced core and HI histone mRNAlevels as determined by hybridization to specifichistone DNA probes. At 4.14xl04kPa for lOmin corehistone and HI histone mRNA levels were reduced32-38% and 58%, respectively. At 30min post-decompression core mRNA levels returned to atmos-pheric control levels while HI histone mRNA levelscontinued to be suppressed. Levels of macromolecu-lar synthesis were monitored under hydrostaticpressure with radioactive precursors of RNA, DNAand protein. Macromolecular synthesis was shown tobe suppressed in a dose-dependent manner withincreasing magnitude and duration of pressure. Todetermine the influence of pressure on histonemRNA stability, actinomycin D (lO/igml"1) was usedto block RNA synthesis. Relative amounts of H4 and

HI mRNA were determined at atmospheric pressureand following treatment with actinomycin D(10/igmT1), pressure (4.14xl04kPa) and a combi-nation of pressure and actinomycin D. This studyshows that a synthesis component and a stabilitycomponent are involved in the pressure-inducedreduction of core histone mRNA. At 4.14x 104 kPa for15min, there was a 42% reduction in core histonemRNA of which approximately one third was due asuppression of transcription and two thirds to a lossof mRNA stability. The pressure-induced reductionin histone mRNA is attributed to the instability ofendogenous histone mRNA and a reduction intranscription/processing of new histone mRNA.

Key words: hydrostatic pressure, histone, mRNA

Introduction

The effect of hydrostatic pressure on cellular systems hasbeen a subject of interest for many years (Zimmerman,1970). A study of hydrostatic pressure-induced pertur-bations of biochemical characteristics of eukaryotic sys-tems may provide an insight into how organisms adapt tohigh pressure as well as a better understanding of generegulation in eukaryotic cells. Several investigators haveshown that the synthesis of macromolecules, in bothprokaryotic and eukaryotic systems, is suppressed follow-ing the application of pressure (for a review, seeZimmerman et al. 1987). One area that has beeninvestigated is the effect of high pressure on eukaryoticand prokaryotic translational events (Scheck and Landau,1982; Schwarz and Landau, 1972; Hermolin and Zimmer-man, 1969). It was found that pressure interferes withprotein synthesis at more than one level. While in someeukaryotic systems such as rabbit reticulocytes, polysomeintegrity was not affected, the integrity of polysomes inother systems such as Tetrahymena pyriformis weredisrupted. Therefore, it is by no means clear how pressureexerts its effects on these systems. Recently, it has beenshown in the protozoan, Tetrahymena pyriformis, thattubulin gene expression is sensitive to pressure (Tahir et

Journal of Cell Science 98, 123-129 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

al. 1988). To gain a better understanding of the influenceof pressure on the sequence of events involved in geneexpression, i.e. transcription/processing and mRNA stab-ility, histone genes in a higher eukaryotic cell line (HeLaS3) were used as a model system for investigation. Anadvantage of using histone genes as a model is thathistone gene expression has been well characterized inthese cells (Stein et al. 1984).

Histones are a set of basic proteins that associate witheach other and with nuclear DNA. In HeLa cells, there arefive types of histones, which can be classified into twogroups, the core histones (H2A, H2B, H3 and H4) and theHI or linker histone. Several workers have shown that therapid onset of histone mRNA levels in S phase is due to twofactors, an increase in the rate of synthesis and an increasein the half-life of the histone message (Heintz et al. 1983;Plumb et al. 1983). Histone gene regulation is also affectedby 3' processing of the nascent histone transcripts. Theregulation of histone biosynthesis is under both transcrip-tional and post-transcriptional control. Thus, we are ableto study the effect of hydrostatic pressure on multiplelevels of gene regulation. In this study we have investi-gated the influence of hydrostatic pressure on macromol-ecular synthesis and histone mRNA levels in HeLa S3cells.

123

Materials and methods

Cell cultureHeLa S3 cells were maintained in suspension culture at 37 °C.Cultures were maintained in suspension-modified Eagle's me-dium (s-MEM) supplemented with calf serum (7%) and L-glutamine (292mgml~1) (Grand Island Biological Company).Cells were used in mid-log growth phase.

Pressure treatmentCells were subjected to pressure treatment for durations of5-30 min at pressures ranging from 6.89 xlO3 to 6.89xl04kPa(1000 to 10000 lbf in"2). Log growth phase cells were centrifugedat 800 # for 5 min, transferred to 15 ml Lucite chambers andcovered with parafilm. The Lucite chambers were placed in astainless steel pressure chamber and connected to the pressurepump. A second Lucite chamber was prepared in a similarmanner and maintained at atmospheric pressure. The experimen-tal (pressure and atmospheric) chambers were placed in atemperature-controlled housing maintained at 37 °C. Hydrostaticpressure was applied at the rate of 1.72x 104 kPa/stroke using anAminco pressure pump (Zimmerman, 1971). At the conclusion oftreatment, pressure was released instantaneously by means of aneedle valve. Cells were removed and placed at 4°C within 30 s ofdecompression. After decompression, macromolecular synthesiswas determined or RNA was isolated. All experiments wereconducted in duplicate with a minimum of three trials. Thestatistical significance of the data was evaluated using Student'st-teet.

Growth dataSamples of cells from a 5-day culture were subjected to4.14xlO'lkPa for 10 min at 37 °C. Following decompression thecells were placed in suspension culture. Cells were counted in ahaemocytometer on a daily basis until stationary growth phase(approximately 6 days). Observations were made with a com-pound microscope fitted with phase-contrast optics.

Macromolecular synthesisMacromolecular synthesis was determined by the incorporation ofradioactive thymidine, uridine and leucine. Samples of cells(5xl06cellsml~1) were removed from suspension culture andcentrifuged at 800^ for 5 min. Cells were resuspended in s-MEMand incubated for 10 min with radioactive precursors, 5//Ciml~1

[3H]thymidine (sp. act. SSCimmol"1), S/zCiml"1 [3H]uridine (sp.act. 45Cimmor1) or 2.5/fCimr1 [3H]leucine (sp. act. 60 Cimmol"1). The incorporation of [3H]thymidine, [3Hluridine and[3H]leucine was used as an index of DNA, RNA and proteinsynthesis, respectively. At 10 min after the initiation of radioac-tive treatment the cells were subjected to hydrostatic pressure ofvarying durations and magnitudes. Upon decompression, thesample was centrifuged at 800 g for 5 min, washed twice withphosphate-buffered saline (PBS) and precipitated with 10 % coldtrichloroacetic acid (TCA). The cell homogenates were filteredthrough a 0.45 /an Millipore filter under low suction. Thehomogenates were washed three times with cold 10 % TCA on thefilter and air dried. The dry filters were then placed in liquidscintillation cocktail containing Permablend I (Packard Instru-ment Co.) and toluene and counted in a Beckman LS 250 liquidscintillation spectrophotometer.

Isolation of total HeLa cell RNAImmediately after decompression, the cells were placed at 4°C,collected by centrifugation and washed twice with PBS. In someexperiments cells were treated with actinomycin D (lO/jgml"1)for 30 min prior to RNA isolation. This concentration was found toblock more than 95 % of RNA synthesis (data not shown). Cellswere resuspended in lysis buffer (2mM Tris-HCl, pH7.4, lmMEDTA, 0.2% sodium dodecyl sulphate (SDS) and l m g m T 1

Proteinase K) and incubated for 15 min at room temperature.Lysates were repeatedly extracted with phenolxhloroform:isoamyl alcohol (25:24:1, by vol.). Nucleic acids were precipitated

with sodium acetate (final concentration 0.25 M) and 3 volumes of95% ethanol at -20°C. The nucleic acids were centrifuged at12 000 # for 30 min and resuspended in TCM buffer (10 mMTris-HCl, pH7.4, 2 min CaCl2, 10 mM MgCl2). The samples weredigested at 37°C for 20min with O.lmgml"1 DNase I that hadbeen previously treated with proteinase K to remove anycontaminating ribonuclease activity (Tullis and Rubin, 1980).The amount of RNA was quantitated at Ajgo and equal amountswere fractionated by electrophoresis on a 1.5% agarose-6%formaldehyde gel. After electrophoresis, the RNA was transferredto nitrocellulose filters and baked in vacuuo for 2 h. For detectionof histone mRNAs, the filters were prehybridized in 50%formamide, 5xSSPE (lxSSPE: 0 .18 M NaCl, 0 .01MNaH2PO4H2O, 1 mM EDTA), 1 % SDS and 1 % Non-fat dried milk(NFDM) (see Baumbach et al. 1987). Prehybridization andhybridization were carried out at 42 °C. The probes for thehistones (pFNC16A (HI), pFF435B (H2), pFF435C (H3),pFO108X (H4)) are DNA clones of human histone genes. Theprobes were nick translated with [32P]dCTP and added to thehybridization mixture (50% formamide, 5xSSPE, 1% SDS, 1%NFDM) at a concentration of lxl06cts min imi" 1 . After hybrid-ization, the filters were washed twice at room temperature in2xSSPE, pH7.7, 0.1% SDS, and twice in lxSSPE, pH7.7, 0.1%SDS. Filters were air dried and exposed to preflashed XAR-5 filmat -80°C. Autoradiographs were quantitated by densitometry.

Results

Morphology and cell growthHeLa cells growing in monolayer on a coverglass retracttheir pseudopodia and assume a round shape whensubjected to hydrostatic pressure of 1.37xl04kPa. Manycells detach from the coverglass or remain loosely attachedto the substratum. With increasing magnitudes of press-ure up to 6.89xl04kPa there is an increase in the numberof cells that become round and detach from the surface.After decompression, cells from monolayer cultures re-attach to the substratum and gradually re-form psuedopo-dia after 1-2 h. Cell viability was not affected by pressure.In contrast, HeLa S3 cells growing in suspension cultureat atmospheric pressure have retracted pseudopodia andare round in appearance, thus hydrostatic pressure doesnot appear to change the shape of these cells whenobserved with phase-contrast microscopy. Nevertheless,following decompression, cells in suspension cultures re-attach to the pressure chamber surface and form pseudo-podia. In general, the effects of pressure on morphology ofHeLa S3 cells are reversible.

Pressure effects on the proliferation of HeLa S3 cellswere determined in cell samples exposed to variousmagnitudes of pressure (6.89XlO3 to 6.89xl04kPa). Thegrowth characteristics of cells in suspension cultures wereevaluated at 4.14xl04kPa, a representative pressure atwhich histone mRNA levels were assessed (Fig. 1). Cellswere subjected to 10 min of pressure treatment(4.14xl04kPa) and placed into a suspension culture; celldensity was plotted as a function of time after decompres-sion. Following pressure treatment, cell proliferation wasinhibited for 2 days, after which the cells returned to anormal growth pattern. The delay in growth observedimmediately after decompression was dependent on themagnitude of pressure and duration of treatment. At1.37xl04kPa for a duration of 10 min, there was a 5-hdelay in growth whereas at 6.89xl04kPa for 10 min, thegrowth delay was approximately 3 days. Increasing theduration of pressure from 10 min to 30 min at4.14x 104 kPa increased the delay in growth from 2 days toapproximately 2i days. Cell viability as assessed by dye-exclusion analysis was not affected by pressure treatment.

124 A. L. Symington et al.

4 6Time (days)

Fig. 1. Growth characteristics of HeLa S3 cells after pressuretreatment in suspension culture. Cells were exposed to4.14xl04kPa for 30min at 37°C (T T). Cells werereturned to suspension culture and samples counted daily.Cells at atmospheric pressure were maintained at 37 °C insuspension culture ( • • ) . Three independent experimentswere performed. Each value represents the mean ± standarderror.

At the highest level of compression and the longestduration of treatment (6.89xl04kPa for a period of30min) cell viability was similar to that observed atatmospheric pressure.

Macromolecular synthesisHydrostatic pressure reduced the level of macromolecularsynthesis in HeLa S3 cells in a dose-dependent manner(Fig. 2). The incorporation of radioactive precursors,[3H]thymidine, [3H]uridine and [3H]leucine was employedas an index of DNA, RNA and protein synthesis,respectively. Cells treated at 4.14xl04kPa for lOminshowed a reduction of 61, 54 and 68% of radioactivethymidine, uridine and leucine, respectively, in the acid-insoluble fraction. The level of macromolecular synthesiswas reduced further (77-80 % of control value) when thelevel of pressure treatment and the duration of treatmentwere increased to the maximum values used in this study(6.89xl04kPa for 30min). These studies provide evidencethat approximately 20 % of the macromolecular synthesisin HeLa S3 cells is able to continue at very high pressure(6.89 xl04kPa for 30min). Pressure effects on macromol-ecular synthesis were reversible; levels of macromolecularsynthesis from pressure-treated cells were equivalent tothose of non-treated atmospheric controls at 30 min post-decompression from 6.89xl04kPa.

Influence of pressure on histone mRNA levelsTo investigate further the effect of pressure on cellularbiosynthesis, histone mRNA levels were studied in cellssubjected to various magnitudes and durations of press-ure. Following pressure treatment, total RNA wasisolated, fractionated on agarose-formaldehyde gels andtransferred to nitrocellulose. The RNA was hybridizedwith histone DNA probes and quantitated by densi-tometry. The reduction of core histone mRNA (H2A, H2B,H3 and H4) and HI histone mRNA levels from controlvalues was dependent upon the magnitude of pressure(Fig. 3). Low magnitudes of pressure (1.37x10* kPa for10 min) reduced core histone mRNA levels by 17 % and HIhistone mRNA levels by 38%. Increasing the level of

10 15 20 25 30Time (min)

50r B

251-C

IOx 20

S 152

a 10

10 15 20Tune (min)

25 30

10 15 20Tune (min)

25 30

Fig. 2. The incorporation of radioactive precursors into theacid-insoluble fraction of HeLa S3 cells. Cells were incubated

3with: (A) [3H]thymidine1 3

(B) [3H]uridine1

[ ] y ( ^ ) ; ( ) [(5/iCimr1); or (C) [3H]leucine (2.5/ri3iml"1)for 10 min prior topressure treatment and then exposed to various magnitudes(up to 6.89xl04kPa) and durations (5-30 min) of pressure at37 °C. Following decompression, cells were washed with coldTCA and radioactivity was determined by liquidspectrophotometry. Protein concentrations of the homogenateswere determined. The graphs represent the results of 4independent experiments. ( • • ) Control. 1.01xl03kPa;(A A) 1.37xl04kPa; ( • • ) 2.74xlO'*kPa; (O O)4.14xl04kPa; (A A) 5.50xl04kPa; (D D) 6.89xl04kPa.

pressure to 6.89xl04kPa for 10 min further reduced coreand HI mRNA levels, to 42 % and 71 %, respectively.While the levels of core histone mRNA are not signifi-cantly different from each other, the levels of HI mRNA

Pressure influences histone mRNA 125

0.6 -

H4

H2A/H2B

a. 4 6lbfin"2, xlO"3

are significantly different from core mRNA levels at1.37xlO4 to 6.89xl04kPa (P<0.01).

Duration of pressure also had a marked effect on histonetranscript levels. Histone transcripts were analyzed inHeLa S3 cells at a pressure of 4.14xl04kPa for varyingdurations. After 10 min, core histone and HI histonemRNA levels were reduced by 32-38% and 56%,respectively; after 30 min core and HI histone mRNAlevels were reduced by 38-47% and 77%, respectively.Thus an increase in exposure to pressure from 10 to 30 minfurther reduced core histone mRNA levels by 6-9%whereas, the level of HI mRNA was suppressed anadditional 24% (Fig. 4). Core histone mRNA levels do notsignificantly differ from each other while the levels of HImRNA are significantly different from core mRNA levelsafter 10 min of pressure treatment (P<0.01). Histone geneexpression is sensitive to the magnitude of compression aswell as to the duration of pressure treatment and HIhistone mRNA is affected by pressure treatment to agreater degree than is core histone mRNA. In addition, thelevel of core histone mRNA tended to plateau after 10 minof treatment, whereas the level of HI histone mRNAcontinued to be suppressed.

Hydrostatic pressure-induced suppression of histonemRNA levels was demonstrated to be reversible. After a30 min recovery period, core histone mRNA levelsreturned to pre-treatment levels (following a 10 mincompression at 4.14xl04kPa); however, HI mRNA levelsremained suppressed by 30 %. These results illustrate thatfollowing decompression, there is complete recovery ofcore histone mRNA whereas HI histone mRNA showsonly partial recovery 30 min after decompression (Fig. 5).

Stability of mRNA transcriptsThe mechanism by which hydrostatic pressure reduces thelevel of histone mRNA may affect any one or all of thethree components that regulate histone mRNA levels. Onecomponent concerns the suppression of histone mRNAsynthesis during pressure perturbation, another com-ponent involves the processing of mRNA and the third

Fig. 3. DNA hybridization analysis of core and HIhistone mRNA from HeLa S3 cells subjected tovarying magnitudes of pressure (up to6.89xl04kPa). Following decompression equalamounts of total RNA were fractionated byagarose-formaldehyde gel electrophoresis andtransferred to nitrocellulose. Followinghybridization with nick-translated DNA probes,autoradiograms were analysed by densitometry. Theinset shows a representative autoradiogramshowing H4 histone mRNA levels at varyingmagnitudes of pressure. C represents atmosphericpressure. The graph depicts a representativeexperiment. Each value is the average of anexperiment conducted in duplicate. Threeindependent experiments were performed. HI(O O); H2A/H2B (A A); H3 ( • • ) ; H4

component concerns the stability of mRNA, in whichpressure perturbation degrades or changes the mRNA insuch a manner that it is no longer able to hybridize withthe histone probes. Since transcription and processingboth occur in the nucleus, we are unable to differentiatebetween these two levels of regulation and therefore inthis study transcription and processing were artificallydesignated as one level of regulation. Thus, histone mRNAlevels are affected by pressure treatment as a consequenceof the (direct or indirect) action of pressure on synthesis/processing of mRNA transcripts and stability of histonemRNA. To distinguish between the effects of pressure onmRNA transcription/processing and mRNA stability,10 jugml-1 actinomycin D was used. At this concentration,actinomycin D suppresses 90 % of RNA synthesis in HeLaS3 cells (see also Gallwitz and Mueler, 1969). While it isnot known whether pressure treatment interferes with thebinding of actinomycin D to DNA, studies have shown thatthe proteins and chemicals (ethidium bromide andproflavin) that intercalate with DNA are not pressure-sensitive (Heremans and Van Nuland, 1977). The sup-pression of histone mRNA synthesis/processing and thedegradation of histone mRNA can be estimated bycomparing levels of histone mRNA in cells followingtreatment with actinomycin D, hydrostatic pressure and acombination of actinomycin D and hydrostatic pressure.Pressure-induced instability was determined by com-paring mRNA levels in cells treated with lOjUgml"1

actinomycin D and cells treated with a combination ofactinomycin D and pressure. The effect of pressure ontranscription/processing was determined by comparinghistone mRNA levels from atmospheric control cells andpressure-treated cells (Table 1). This relationship can beexpressed as:

where, Pe is the total pressure effect; P; is the inducedinstability, that is the difference between cells treatedwith actinomycin D and those treated with actinomycin Dand pressure; Pt is the pressure effect on transcription/processing of histone mRNA. This represents the differ-

126 A. L. Symington et al.

0.6 -

co

1 0.4o

0.2 "

Time (min)5 10 15 20 30 C

H1

10 15 20Time (min)

25 30

Fig. 4. The relative amounts of histone mRNAfollowing pressure treatment of 4.14x10* kPa forvarying durations of time. Equal amounts of totalRNA were fractionated by gel electrophoresistransferred to nitrocellulose and hybridized to nick-translated DNA probes. The amount of histonemRNA was detected by densitometry of theautoradiograms. The inset is a representativeautoradiogram showing HI histone mRNA levels atvarious durations of compression. The graph depictsa representative experiment. Each value is theaverage of experiments conducted in duplicate.Three independent experiments were performed. HI(O O); H2A/H2B (A A); H3 ( • •) ; H4

ence between the total pressure effect on histone mENAand the effect of pressure on mRNA stability.

The level of H4 mRNA was reduced by approximately42% after pressure treatment of 4.14xl04kPa for 15 min.Of this value 16% was due to a suppression intranscription/processing and 26% was attributed to areduction in mRNA stability. The other core histones(H2A, H2B, H3) showed a similar suppression as a resultof pressure perturbation. HI mRNA showed a greatersensitivity to pressure perturbation than the core his-tones. HI histone mRNA was reduced by approximately72 % after 15 min at 4.14x10" kPa. Of this value, 38 % wasdue to a reduction in transcription/processing and 34%was attributed to a reduction in mRNA stability (Fig. 6).Since this method provides an indirect determination ofthe effect of pressure on mRNA transcription/processing,a more direct method of assessing transcription/processing is that of investigating in vitro transcriptionusing isolated nuclei (Greenberg and Ziff, 1984). Theseexperiments are in progress.

Discussion

In general, these studies demonstrate that hydrostaticpressure reduces the levels of macromolecular synthesisand histone mRNA. The pressure-induced reduction inhistone mRNA is attributed to the instability of endogen-ous histone mRNA and to a reduction of transcription/processing of new histone mRNA. These studies supportand extend our earlier reports (Tahir et al. 1988) on theinfluence of hydrostatic pressure on tubulin synthesis indeciliated Tetrahymena. The reversibility of pressureeffects on cell morphology, growth and biochemical eventsin HeLa S3 cells was similar to that reported in protozoa,marine eggs and cultured cells (Zimmerman et al. 1987).Under the specified pressure parameters (6.89 xlO3 to6.89xl04kPa for 30 min), cell viability is not affected bypressure treatment although proliferation was delayed.Cell morphology and growth patterns change in responseto pressure and cells resume normal shape and growthpatterns after decompression.

J3o

S 0.1

10 20 30

Time (min)

Fig. 5. HI and core histone mRNA fromHeLa S3 cells treated for 10 min at 4.14X104

kPa at 37 °C and allowed to recover atatmospheric pressure. At times followingdecompression cell samples were removed andRNA was isolated. Equal amounts of RNAwere fractionated by agarose-formaldehydegel electrophoresis, transferred tonitrocellulose and hybridized to nick-translated DNA probes. Autoradiograms wereanalysed by densitometry. The graph depictsa representative experiment. Each value isthe average of experiments conducted induplicate. Three independent experimentswere performed. HI (O O); H2A/H2B(A A); H3 ( • • ) ; H4 ( • • ) .

Pressure influences histone mRNA 127

Table 1. Relative amounts of H4 and HI mRNA at atmospheric pressure and following treatment with actinomycinD, pressure and combinations of pressure and actinomycin D

A. Relative amount of histone H4 mRNA

Time(min)

Atmosphericpressure (l.OlxlO3 kPa)

Pressure(4.14xl04kPa)

Actinomycin D(lO/igmP1)

Actinomycin D+pressure

0510152030

0.640.620.650.620.640.61

0.600.500.420.370.360.32

0.620.440.400.350.320.30

0.590.360.250.180.120.06

B. Relative amount of HI histone mRNA

Time Atmospheric Pressure Actinomycin D Actinomycin D(min) pressure (1.01 xl03kPa) (4.14X104 kPa) (10/igml"1) +pressure

0510152030

0.540.560.550.540.560.53

The time represents the duration of treatment.

0.530.310.230.180.160.14

0.530.440.400.360.300.20

0.540.350.250.190.110.09

H4 HI5

H4 HI10

Time (min)

H4 HI15

Fig. 6. DNA hybridization analysis of HI and H4 histonemRNA from cells treated with pressure, actinomycin D or acombination of actinomycin D and pressure. The amount oftotal suppression is a function of both pressure-inducedinstability of histone mRNA (•) and pressure-inducedsuppression of transcription (stippled). Three independentexperiments were performed. Each value represents the mean.

Core histone mRNA and HI histone mRNA levels werelowered following pressure perturbation. HI histonemRNA acts independently of the core histone mRNAs(H2A, H2B, H3 and H4), which remain coordinatelyregulated in their response to magnitude and duration ofpressure treatment. In addition, in all the studies HIhistone mRNA was more sensitive to pressure treatmentthan core histone mRNA. At both low (1.37xl04kPa) andhigh (6.89xl04kPa) pressure, the.HI mRNA levels weresuppressed by approximately twice that of the core histonemRNA levels. It is possible, therefore, that different

regulatory mechanisms are concerned with the synthesisof Hi and core mRNA, which may account for thedifferential sensitivity to pressure perturbation. Analternative explanation for the greater pressure sensi-tivity of HI histone mRNA is that HI histone mRNA maybe less stable to pressure perturbation than core mRNA. Itis of interest to note that at moderate pressure(4.14 x 104 kPa for 10 min) DNA synthesis is suppressed by61 % while core histone levels are suppressed by 34-48 %.It is possible, though highly speculative, that pressuredissociates the dependence of DNA replication and corehistone mRNA levels.

Our studies show that at least two components(transcription/processing and stability) are involved inthe pressure-induced reduction in histone mRNA. Forexample, there was a 40 % reduction in the level of H4mRNA after pressure perturbation (4.14xl04kPa for15 min) of which approximately one third was due to asuppression in transcription/processing while two thirdswas due to a loss in mRNA stability. Other core histonemRNAs were similar to the H4 fraction with respect tototal reduction in histone mRNA and the relative amountsof suppression attributed to transcription/processing andstability. However, the total reduction in histone HImRNA and the relative amounts of suppression attributedto transcription/processing and stability were different.HI histone mRNA levels were reduced by about 72 % afterexposure to 4.14xl04kPa for 15 min and approximatelyhalf of the suppression was attributed to a reduction intranscription/processing and half to a reduction instability. Therefore, it appears that HI mRNAtranscription/processing is more sensitive to pressureperturbation than H4 mRNA transcription/processing.Several workers have shown that different transcriptionfactors are involved in regulation of the different histonegenes (La Bella et al. 1989; Dailey et al. 1988; Fletcher etal. 1987). It is possible, therefore, that the Hl-specifictranscription factor is more sensitive to pressure thanthose transcription factors that are specific to the corehistone genes. The differential sensitivity of histone HImRNA may be related to the specific structural role of thelinker histone (HI) in chromatin assembly. Histone HI is

128 A. L. Symington et al.

thought to play an active role in chromatin activity, cellproliferation and DNA replication (Sun et al. 1989).

These studies show that HeLa S3 cell proliferation isvery sensitive to hydrostatic pressure, since a short(lOmin) pulse of pressure (4.14xl04kPa) resulted in a 2-day delay in the initiation of cell proliferation. In additionthe delay of cell proliferation was a function of themagnitude of pressure and the duration of treatment.Since histone mRNA levels and macromolecular synthesisare at their lowest level immediately after decompressionwe considered the possibility that the pressure-inducedreduction of histone mRNA and macromolecular synthesisplayed a regulatory role that resulted in delay ofproliferation. We conclude, however, that this assumptionis doubtful and we do not believe that the reduction inhistone mRNA levels and macromolecular synthesisplayed a regulatory role in these experiments, since bothevents returned to pre-pressure levels within l h afterdecompression. The delay in proliferation is more subtle,possibly acting through a cytoskeletal disruption thatlinks translational events with the cytoarchitecture.

In previous studies (Tahir et al. 1988) we have shown areduction in tubulin mRNA in response to pressuretreatment. Since similar results were obtained in thecurrent studies it is possible that comparable mechanismsmay be affected by pressure in both systems and thatpressure affects the regulation of both transcriptional andpost-transcriptional mechanisms. Zambetti et al. (1985)have shown that histone mRNA is preferentially associ-ated with the cytoskeleton. In addition, Bagchi et al. (1987)have shown that polysomal mRNPs are predominantlyassociated with the cytoskeleton, suggesting that thisassociation may have some role in post-transcriptionalregulation. In view of the fact that pressure has beenshown to disrupt native myosin filaments in vitro as wellas cytoskeletal elements such as microtubules and actinfilaments (Salmon, 1975; Swezey and Somero, 1985;Bourns et al. 1988; Tumminia et al. 1989), it is likely thatpressure destroys the association of the mRNA with thecytoskeleton by disorganizing cytoskeletal structure. Ifthis mRNA-cytoskeleton association confers stability onthe mRNA, then any disruption would cause a loss ofstability. In addition, Peltz and Ross (1987) have suggestedthat another mechanism of post-transcriptional regu-lation is the autogenous regulation of histone geneexpression by free histone proteins; they reported that asthe concentration of free histones in the cytoplasmic poolsincreases, histone mRNA stability decreases. Data tosupport this proposal have been reported by otherinvestigators (Hereford et al. 1981; Stein and Stein, 1984).We are at present investigating the effect of pressure onhistone pools and on cytoskeletal associations withmRNA.

This study was supported by NSERC Canada. A.L.S. wassupported by a NSERC postgraduate scholarship.

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(Received 4 May 1990 - Accepted, m revised form, 18 October 1990)

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