Virus Effects of Protease Inhibitors on Chemical ...

6 downloads 0 Views 1MB Size Report
Aug 1, 1979 - inhibitors, leupeptin and antipain, can reduce the induction of type C virus by inhibitors of protein synthesis, suggesting a role for proteolysis in ...
Effects of Protease Inhibitors on Chemical Induction of Type C Virus Cedric W. Long, Joan A. Bruszewski, Wayne L. Christensen, et al. Cancer Res 1979;39:2995-2999. Published online August 1, 1979.

Updated Version

E-mail alerts Reprints and Subscriptions Permissions

Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/39/8/2995

Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected].

Downloaded from cancerres.aacrjournals.org on July 9, 2011 Copyright © 1979 American Association for Cancer Research

[CANCER RESEARCH 39, 2995-2999, August 1979]

Effects of Protease Inhibitors on Chemical Induction of Type C Virus' Cedric W. Long,2 Joan A. Bruszewski, Wayne L. Christensen, and William A. Suk Biological Carcinogenesis Program, Frederick Cancer Research Center, Frederick, Maryland 2 1701

ABSTRACT A role for proteolysis during chemical induction of endoge nous xenotropic Type C virus from Kirsten sarcoma virus transformed mouse cells was examined. Two distinct classes of protease inhibitors, the trypsin inhibitor, a-N-tosyl-L-lysine chloromethyl ketone, and two naturally occurring oligopeptide inhibitors, antipain and leupeptmn,were found to inhibit induc tion of virus by cycloheximide and histidinol. Virus activation by 5-iododeoxyuridine was inhibited to a lesser degree. During the time cells were exposed to these compounds, there was little inhibition of [3H]uridine incorporation into total cellular RNA or polyadenylic acidt cytoplasmic messenger RNA, sug gesting that inhibition of proteolysis, and not RNA transcription, was responsible for blocking virus induction. INTRODUCTION Several studies have shown that treatment of cells with chemical or physical agents can cause latent virus genomes to be temporarily or permanently activated. In many cases, the same agents can induce a variety of viruses from different cells with varying degrees of effectiveness. Thus, halogenated py rimidines (IdUrd and 5-bromodeoxyuridine) and inhibitors of protein synthesis (cycloheximide and puromycin) have all been shown to activate polyoma virus from rat cells (13, 29), SV4O virus from hamster cells (24, 25), Epstein-Barr virus from human lymphoblastoid cells (14, 21 , 22), and type C viruses from murine cells (1 , 3, 28). Furthermore, in some of these systems, mitomycin C (13, 25), UV (13, 25), hydroxyurea (34), X-irradiation (13, 41 ), amino acid deprivation (22, 24), and analogs of amino acids (4, 27) have also been shown to be effective inducers. Although many diverse means of activating these viral genomes have been found, the discrete biochemical steps occuring during each process have not been identified, and a mechanism remains obscure. Perhaps the most intensively studied and best understood example of virus activation involves induction of A phage. In this system, treatment of lysogenic bacteria with mitomycin C or UV irradiation results in production of phage (8). Following physical or chemical treatment, the specific DNA-binding activ ity of phage repressor disappears

from the induced bacteria

(37). More recently, it has been demonstrated that the A re 1This work was supported

by Contract

NOl -CO-75380

with the National

Cancer Institute, NIH, Bethesda, Md. 20205. 2 To

whom

requests

for

reprints

should

be

addressed,

at

Frederick

Cancer

Research Center, P.O. Box B, Building 560, Frederick. Md. 21701. 3 The

abbreviations

used

are:

IdUrd,

5-iododeoxyuridlne;

TLCK,

N-a-tosyl-L

lysine chronomethyl ketone; K-BALB, Kirsten sarcoma virus-transformed BALB/ 3T3 cell line; FRE. fetal rat embryo cells; MEM, Eagle's minimal essential medium; TCA. trichloroacetic acid; SDS. sodium dodecyl sulfate; poly(A), polyadenylate;

poly(A)@ mRNA, polyadenylate containing cytoplasmic messenger RNA; oligo(dT), oligodeoxythymidylate; PBS, phosphate-buffered saline [CaCI, (100 @og/mI)-KCl (200 @g/mI)-KH2PO4; (200 @g/ml)-MgSO4(59.2 og/mI)-NaCI (800 og/mI)-NaHPO4(1 150 sg/mI)]. Received November 17, 1978; accepted April 26, 1979.

pressor is proteolytically cleaved both in vivo and in vitro when bacterial lysogens are induced by UV or mitomycin C treatment (35) and that the proteolytic inactivation of repressor and induction of phage can be prevented by treatment of cells with antipain, an oligopeptide protease inhibitor (30). In an effort to understand the mechanisms involved in type C virus activation, we examined the effect of various protease inhibitors on the induction process. Here we report findings which demonstrate that the trypsin inhibitor, TLCK, as well as 2 oligopeptide inhibitors, leupeptin and antipain, can reduce the induction of type C virus by inhibitors of protein synthesis, suggesting a role for proteolysis in induction. MATERIALS AND METHODS Cells. K-BALB 19a cellswere clonedfroma Kirstensarcoma virus-transformed BALB/c 3T3 cell line (K-BALB), originally obtained from Dr. S. A. Aaronson (National Cancer Institute, Bethesda, Md.), and chosen for virus induction studies because of high levels of type C RNA virus activation (5). This clone was used for studies concerning TLCK inhibition. For experiments dealing with permeabilization, 16c, a highly inducible, relatively flat clone (5), was used to avoid detachment of cells during the hypertonic salt treatment. FRE were obtained from Dr. E. Scolnick, NIH. All cells were grown in MEM containing 10% fetal calf serum, 100 lU penicillin, and streptomycin (100 @&g/ ml). All cells were tested and found to be free of Mycoplasma contamination in tests performed by R. Del Giudice (Frederick Cancer Research Center). Chemicals were from the following commercial sources: TLCK, a-N-tosyl-L-phenylalanine chloro methyl ketone, phenylmethylsufonyl fluoride, cycloheximide, histidinol, IdUrd, actinomycin D, and mitomycin C were from Calbiochem, San Diego, Calif.; N-a-tosyl-L-arginine methyl es ter and dexamethasone were from Sigma Chemical Co., St. Louis, Mo. Antipain and leupeptin were the generous gifts of Dr. H. Umezawa. Induction of Virus. The assay for virus induction was a modification of the procedure described by Aaronson and Dunn (2). K-BALB 19a cells were plated at 5 x 1O@cells/6-cm dish in MEM containing 5% fetal calf serum 5 to 7 days before induction. To increase virus activation, cells partially synchro nized were released from the serum-starved state with MEM containing 20% fetal calf serum I 6 to 18 hr before induction (20).

The

induction

medium

used

was

MEM

containing

anti

biotics, 10% fetal calf serum, dexamethasone (0.1 @g/ml)to enhance virus production, and the chemical inducer (12). When an amino acid analog was used as an inducer, the analogous amino acid was omitted from MEM, and dialyzed fetal calf serum was used. The cells were incubated in inhibition and/or induction medium at 37°for the specified time, washed 3 times with PBS, and incubated in the dark in medium containing mitomycin C (20 jtg/ml). After 1 hr, the mitomycin C-containing medium was removed, the cells were washed 3 times with PBS and trypsinized, and 5 x 1O@ cells were plated into 6-cm dishes seeded the previous day with 1.5 x 1O@FRE cells in Polybrene

AUGUST1979

2995

Downloaded from cancerres.aacrjournals.org on July 9, 2011 Copyright © 1979 American Association for Cancer Research

C. W. Long et a!. (2 @g/ml).Simultaneously,3.5 x 1O@ treated or untreatedcells were plated in MEM into 6-cm dishes. The next day, these cells were trypsinized and counted to determine viability. Foci were counted 7 to I 0 days later. Macromolecular Synthesis. The synthesisof RNA or DNA was measured by the amounts of radioactive uridine or thymi dine incorporated into TCA-insoluble material. K-BALB 19a cells were treated with the protease inhibitors for the specified time and incubated with either [G-3H]uridine (1 @tCi/ml)(New England Nuclear; 3.6 Ci/mmol) or[methyl-3H]thymidine (1 zCi/ ml) (New England Nuclear; 6.7 Ci/mmol). After 30 mm, the radioactive medium was removed, and the cells were washed twice with cold PBS. Each dish was washed with two 2-mI aliquots of cold PBS plus 0.5% SDS and precipitated with 10% TCA.

The

precipitates

were

collected

on

glass-fiber

@

using

the

phenol

chloroform

method

of

Penman

RESULTS

filters,

washed twice with 5 ml of cold 5% TCA, washed once with 5 ml of 2-propanol, dried under a heat lamp, and counted in liquid scintillation fluid. Simultaneously, macromolecular syn thesis was measured in untreated controls. All values were determined in triplicate. RNA Isolation. Duplicate cultures of each treatment were washed and prepared for RNA extraction according to the procedure of Johnson et a!. (23). The extraction was done at 550

NaCI in 100 ml of serum-free MEM were added to each dish. Permeability was monitored by uptake of trypan blue. Approx imately 60 mm were required for >80% of the cells to stain, after which the hypertonic medium was removed by washing twice with 4 ml of serum-free MEM. The resealing process was initiated by adding MEM-10% fetal bovine serum and monitored by dye exclusion. Generally, 3 hr were required for >80% of the cells to show no dye uptake. At this time, the monolayer was washed twice with PBS, and chemical induction was carried out for 16 hr. When the protease inhibitors antipain and leupeptin were tested, they were included during the hypertonic salt treatment and the subsequent resealing period.

(31

).

The

RNA extracted into the aqueous phase was combined with 2.5 volumes of 100% ethanol and left overnight at —20°. The ethanol-precipitated RNA was centrifuged at 11,500 x g for 1 hr, and, depending on the treatment, 45 to 65% of the extracted radioactivity could be pelleted. The poly(A)―mRNA was isolated using oligo(dT) cellulose (38). Fifteen-cm columns were packed with 0.8 ml of oligo(dT) cellulose (type 2; Collaborative Research, Waltham, Mass.) and equilibrated with elution buffer [10 mMTris (pH 7.4)-0.05% SDS]. To eliminate nonspecific binding, I 00 @g of yeast tRNA (Sigma Chemical Co.) were washed through the column, fol Iowed by an elution buffer rinse. The column was then equili brated with binding buffer [400 mM NaCl-10 mM Tris (pH 7.4)0.5% SDS], and the sample, dissolved in 1 ml of binding buffer, was then applied. Four ml of binding buffer were passed through the column to elute the rRNA and tRNA. This was followed by 2 ml of elution buffer to obtain the poly(A)@mRNA. A 10-@tlaliquot of each fraction was counted to determine the incorporation of [3H]uridine into this RNA species. The first 4 fractions eluted with the binding buffer (rRNA and tRNA) were combined and precipitated with 2.5 volumes of ethanol. The RNA was again centrifuged at I I .500 x g, resuspended in SDS buffer [0.1 M NaCl-0.01 M Tris (pH 7.4)-0.001 M EDTA 0.5% SDS], and layered onto 15 to 30% sucrose gradients. The gradients were centrifuged using a Beckman type SW27 rotor for 17 hr at 24,000 rpm and 24°.Each gradient was fractionated by pumping from the bottom through a 20-id micropipet to a UV monitor and then collected in 1.25-mI fractions. A 0.5-mI aliquot of each was counted to determine the incorporation of [3H]uridine into the RNA species. Preparation of Permeable Cells. Permeabilizationwas car ned out according to the method of Castellot et a!. (1 1) with the following modifications. Cells to be permeabilized were plated at a density of 2 x 10@cells/6-cm dish in MEM con taming 5% calf serum. After 24 hr. the medium was removed, and the cells were washed 4 times with 3 ml of PBS. The wash was removed and 2.5 ml of a solution consisting of 3.4 g of

The role of proteolysis in chemical induction of type C virus was studied by testing the inhibitory properties of several synthetic and naturally occurring protease inhibitors on virus activation. The synthetic compounds included TLCK, N-a-tosyl L-arginine methyl ester, a-N-tosyl-L-phenylalanine chloro methyl ketone, and PMSF. Because the synthetic compounds contain highly reactive groups that could react with cell con stituents other than proteases, brief treatment times and con trols to exclude nonspecific effects were necessary. To study the action of these chemicals on virus activation, K-BALB cells were induced with either cycloheximide, histidinol, tyrosinol, or IdUrd in the presence or absence of protease inhibitors for 4 hr and plated onto FRE cells for focus formation. Of the compounds tested, TLCK, a trypsin inhibitor, very efficiently inhibited virus activation. During a 4-hr incubation period, more than an 80% decrease in virus activation by inhibitors of protein synthesis was observed (Chart 1). IdUrd induction, on the other hand, was reduced somewhat less, usually about 50% during 4 hr of incubation. The greatest inhibition of induction occurred when TLCK was present during the incubation of cultures with chemical inducers, suggesting blockage of a proteolytic event during and not after induction (Table 1). Less inhibition was found when TLCK was added to cultures and removed before

C 0 U ‘Q C 0 C 0

C) 0

TLCK

@glmI

Chart 1. K-BALB 19a cells were released from a serum-starved state; 16 to 18 hr later, variable concentrations of TLCK were added to cultures in MEM 10% fetal bovine serum. Thirty mm later, the cultures were induced with IdUrd (30 @g/ml),cycloheximide (25 pg/mI), histidinol (500 gig/mI), and tyrosinol (500

@og/ml) for 4 hr in the presence of TLCK. Medium was then removed, and cells were assayed for foci on FRE monolayers as described in “Materials and Methods.― The control number of focl/5 x 10@K-BALB cells plated was 1166 for IdUrd, 685 for cycloheximide, 813 for histidinol, and 649 for tyrosinol.•, IdUrd; 0, cycloheximide; t@,histidinol; A, tyrosinol.

CANCERRESEARCHVOL. 39

2996

Downloaded from cancerres.aacrjournals.org on July 9, 2011 Copyright © 1979 American Association for Cancer Research

Protease Inhibitors and Type C Virus Induction

induction or when cultures were induced and subsequently exposed to TLCK. Maximum inhibition was found when TLCK was added 30 to 40 mm prior to and during the 4-hr induction interval. The other synthetic protease inhibitors were capable of inhibiting activation, but concentrations 5- to 50-fold greater than those with TLCK were necessary, and cytotoxicity was regularly observed. In the case of TLCK, cell viabilities, as measured by trypan blue exclusion, remained above 90%, and plating efficiencies were reduced less than 10%. However, TLCK in excess of 100 @sg/mI reduced plating efficiencies more than 50% following a 4-hr incubation. Two types of studies were carried out to exclude nonspecific inhibition of cellular RNA or DNA synthesis by TLCK as the mechanism of blocking virus activation. K-BALB cells were treated with TLCK (50 @g/ml)for 1, 3, and 5 hr followed by 30mm pulse with either [3Hjuridine or [3Hjthymidine. Under these conditions, TLCK reduced RNA and DNA synthesis by less than 50%, whereas cycloheximide, an inducer, reduced RNA synthesis by 75% after 5 hr and almost completely shut down DNA

synthesis

after

1 hr (Chart

2).

Since

type

C viral

TLCK

was

found

to reduce

synthesis

by about

5%,

while

cycloheximide and actinomycin D reduced incorporation by 60 to 90%, respectively (Table 2). There was no apparent reduc tion in uridine incorporation into rRNA. As a part of this exper iment, to ensure that TLCK was effectively blocking induction, duplicate dishes were induced with cycloheximide or IdUrd in the presence of TLCK (50 pg/mI), and virus activation was determined by focus formation on FRE monolayers. In both cases, activation of focus-forming virus was reduced approxi mately 85 to 95%. Recently, several naturally occurring protease inhibitors pro duced by actinomycetes have been isolated and characterized (6).

These

inhibitors

are

aldehyde

derivatives

of oligopeptides

Table 1 Time-dependent andafter K-BALB

1 9a cells were

released

TLCK inhibition from a serum-starved

state,

@g/ml) 16 to 18 hr they wereinducedwith eithercycloheximide(25 timesat or histidinol(500 @zg/ml). TLCKwas addedat the indicated asdescribed.Treatment 50 @ig/ml.Treated cells were assayedfor foci on FRE cells time Focl/5 x trol8Cycloheximide 3 hr

3 hr

100Cycloheximide 24+

10@K-BALB

Cycloheximide Cycloheximide

261 65

Cycloheximide 78+

204

None None

149 36

TLCK

118

100TLCK

Cycloheximide

177

45a

Cycloheximide

80

TLCKCycloheximide

TLCKCycloheximide 100Cycloheximide 24+ TLCKCycloheximide 79None

Control

Induction

refers

to absence

of TLCK.

0

a 0 U C 0 C 0

C)

..-@

3 5 Time . Hours Chart 2. K-SALB 19a cells were released from serum-starved state; I 6 to 18 hr later they were treated with cycloheximide (25 pg/mI) and TLCK (50 pg/mI) for 1, 3, or 5 hr. Following the period, [3H)urldlne or [3H)thymldlne Incorporation was carried out as described In ((Materials and Methods. ‘ 0. t@,0, [3H]urldine Incorporation; •,A, •,[3H]thymldlne incorporation. 0, •,control cultures; t@, A, TLCK (50 pg/mI);

0,

@,cycloheximide

(25 pg/mI).

RNA

contains sequences of poly(A) (15, 19, 26), a second means to exclude a TLCK-mediated transcriptional block involved testing its effect on poly(A)-containing cytoplasmic mRNA syn thesis. Accordingly, K-BALB cells were treated for 4 hr with TLCK (50 @tg/ml),and [3H]undinewas pulsed into cells for 90 mm in the presence of TLCK. Poly(A)-containing RNA was isolated from cell extracts by oligo(dT) cellulose chromatogra phy.

C 0

% of con

Table2 Effect of inhibitors on poly(A) mRNA synthesis

Two x 1O@K-BALB I 9a cells were plated into 10-cm dishes in MEM-10% fetal bovineserum.After 72 hr. the mediumwas changed, and 16 hr later, cultureswereexposedto chemicalsfor 4 hr followed by a 90-mm labeling period with [3H]undinein the presenceof the inhibitory chemicals.The cultures were then processedfor RNA as described

in “Materialsand Methods. incorporationControl100TLCK Inhibitor%

[3H]uridlne

pg/mI)95Cycloheximide (50

pg/mI)39Actinomycin (25 D (1 .2 pg/mI)1

1

and are advantageous for biological studies because they do not contain highly reactive groups capable of reacting with cell constituents, are relatively nontoxic to cultured cells, and have low molecular weights. Preliminary experiments with K-BALB 19a cells showed no inhibition of induction when leupeptin (1000 @g/ml)was included during a 4-hr treatment with chem ical inducers. Since the ability of these compounds to penetrate intact cells was uncertain, a reversibly permeabilized K-BALB cell system was developed to more carefully examine their action on induction. Permeabilization and chemical induction were carried out sequentially on K-BALB cells in dishes without intermediate trypsinization. It was found that a highly inducible, relatively flat variant, K-BALB 16c, was best suited for the experiments since it was not appreciably affected by the hy pertonic salt treatment, showed little detachment, and resealed almost completely after 3 hr as measured by dye exclusion. Following this treatment, virus activation during a 16-hr induc tion varied between 20 to 80% of nonpermeabilized cultures. Leupeptin and antipain (50 to 200 zg/ml) did not reduce the level of virus induction by IdUrd or cycloheximide in nonper meabilized cells (Table 3). However, both compounds did re duce activation when introduced during the permeabilization and resealing periods. IdUrd induction was reduced 20 to 30%, while cycloheximide activation was inhibited 80% by 200-@sg/ ml concentrations of each oligopeptide. Viabilities as deter mined by dye exclusion remained high in each case. When cells were induced with cycloheximide for 20 hr and subse quently permeabilized and resealed in the presence of protease

AUGUST1979

2997

Downloaded from cancerres.aacrjournals.org on July 9, 2011 Copyright © 1979 American Association for Cancer Research

C. W. Long et a!. Table3 Effect ofleupeptin and antipain on virus inductionInducer and InhibitorFoci/S tionaViabilitiesNonpermeabilizedbIdUrd

of con x trol induc 10@cells%

charge, and growth conditions (36). It has been well docu mented that abnormal proteins are degraded much more rap idly than are their normal counterparts. Thus, mutant forms of !ac repressor (32), $-galactosidase

(1 8), and mammalian hy

poxanthine-guanine phosphoribosyltransferase (10), as well as abnormal amino acid analog containing proteins or fragments of proteins produced by treatment with puromycin, turn over 00 IdUdr + 50 @og antipain 713 97 105 very rapidly, perhaps through exposure of proteolytically sen IdUrd + 200 pg antipain734 8261 11296 95IdUrd sitive sites (16). Recently, it has been shown that a proteolytic + 50 pg leupeptin 17 event is involved in A phage activation. In this system, exposure IdUrd + 200 pg leupeptin863 8551 11698 112Cycloheximide of bacteria to UV, mitomycin C, or other agents that cause DNA damage results in coordinate expression of several functions 00 Cycloheximide + SOpg antipain 400 83 78 including A phage, collectively known as SOS functions (33, Cycloheximide + 200 pg antipain484 4011 77Cycloheximide 8385 39). Following physical or chemical treatment, the DNA-binding activity of phage repressor disappears from the bacteria + 50 pg leupeptin Cycloheximide + 200 pg leupeptin476 55198 11470 72Permeabilized@'IdUrd through proteolytic cleavage to an inactive form (35), allowing phage expression to proceed. The induction of SOS functions and A repressor inactivation can be blocked by addition of the protease inhibitor antipain, to bacteria during induction treat IdUrd + 50 1@g antipain 51 66 190 ment (30). IdUrd + 200 pg antipain77 47100 61136 210IdUrd Although a precise mechanism for induction of type C viruses + 50 pg leupeptin 19 54 from cells is not known, several studies indicate that a possible IdUrd + 200 pg leupeptin1 711 9268 67Cycloheximide model might involve disruption of DNA regulatory protein com 9 00 plexes and enhanced transcription of the viral genome. For Cycloheximide + 50 pg antipain 110 34 208 example, it has been shown that IdUrd must be incorporated Cycioheximide + 200 pg antipain31 391 1294 205Cycloheximide into DNA for it to induce. Chemicals that block DNA synthesis + SOpg leupeptin inhibit IdUrd induction (40). Induction by both halogenated Cycloheximide + 200 pg leupeptin72 3823 1283 97PermeabilizedCCycloheximide pyrimidines and inhibitors of protein synthesis require de novo cellular RNA synthesis and can be blocked by actinomycin D (9). During the activation by cycloheximide and IdUrd, viral 93 00 RNA accumulates in both nucleus and cytoplasm, suggesting Cycloheximide + 75 pg antipain 181 94 ND Cycloheximide + 75 pg leupeptin1 2681 138NDd ND that these chemicals cause a transcriptional increase in viral a Control refers to absence of protease inhibitor. RNA (7, 9). The present study demonstrates that protease b K-BALB 1 6c cells were plated at 2 x 1 O@cells/6-cm dish in MEM-S% calf inhibitors can block the chemical induction of endogenous serum. After 24 hr. medium was removed, and cells were washed 4 times with 3 ml of PBS. The cells were permeabilized as described in ‘ Materials and Methods'S xenotropic virus from mouse cells. The induction by protein and resealed for 3 hr. Protease inhibitors were included during hypertonic salt synthesis inhibitors was more sensitive to these chemicals than treatment and subsequent resealing. Chemical induction was carried out for 16 induction by IdUrd. In a model involving DNA-regulatory protein hr followed by mitomycin C treatment and overlay onto FRE cells for focus formation assay. Nonpermeabilized cells were treated with protease inhibitors interactions similar to the A phage system, halogenated pyrim for 4 hr, a time corresponding to permeabilization and resealing. idine incorporation into DNA could be enough to form altered C K-BALB 1 6c cells were plated as above, and after 1 6 hr, they were induced with cycloheximide for 20 hr. Following this treatment, cells were washed, conformations and prevent repressor binding. However, if syn permeabilized, and resealed as above in the presence or absence of antipain or thesis of a regulatory protein was inhibited by cycloheximide, leupeptin. The induced, nonpermeabilized control gave 767 foci. previously formed protein would presumably still exist depend d ND, not done. ing on turnover degradation rates and could still repress virus synthesis. It is in the latter instance that naturally occurring inhibitors, no reduction in activation of virus was observed, proteolysis would be necessary to inactivate a regulatory pro suggesting that inhibition was unique to a process occurring tein, allowing transcription and activation of the viral genome during the induction interval. When introduced into permeabil to occur. Although a mechanism of induction is far from for ized cells, these protease inhibitors did not reduce subsequent mulation, the identification of separate events that occur during incorporation of uridine and thymidine into cellular RNA and induction by different chemicals may eventually clarify the DNA. regulatory process that operates in controlling virus expres sion. The availability of a permeabilized and inducible cell DISCUSSION system should provide means of testing many biologically active compounds which normally penetrate cells poorly. Protein degradation has become recognized as a biologically and clinically important regulatory process in a number of REFERENCES diverse systems (16, 17, 36). In both prokaryotic and eukary otic cells, degradation through proteolysis is a continual and 1. Aaronson, S. A., and Dunn, C. Y. High frequency C-type virus induction by variable process whereby cells can respond to changes in the inhibitors of protein synthesis. Science 183: 422—424,1974. 2. Aaronson, S. A., and Dunn, C. Y. Endogenous C-type viruses of BALB/c environment and maintain a proper balance of individual cel cells: frequencies of spontaneous and chemical induction. J. Virol., 13: lular proteins. Degradation rates of individual cellular proteins 181-185, 1974. are highly variable and can depend on such factors as size, 3. Aaronson, S. A., Todaro, G. J., and Scolnick, E. Induction of murine type C 2998

CANCER

Downloaded from cancerres.aacrjournals.org on July 9, 2011 Copyright © 1979 American Association for Cancer Research

RESEARCH

VOL. 39

Protease Inhibitors and Type C Virus Induction viruses from clonal lines of virus-free BALB/3T3 Cells. Science, 174: 157— in resting and growing cells. Cell, 1: 95—100,1974. 159, 1971. 24. Kaplan, J. C., Wilbert, S. M. , and Black, P. H. Analysis of simian virus 40 4. Aksamit, R. R., Christensen, W. L., and Long, C. W. Characterization of type induced transformation of hamster kidney tissue in vitro. VIII. Induction of C virus activation of amino acid analogs. Virology, 83: 139—1 49, 1977. infectious simian virus 40 from virogenic transformed hamster cells by amino 5. Aksamlt, R. R., and Long, C. W. Isolation of thioguanine resistant variants of acid deprivation or cycloheximide treatment. J. Virol., 9: 448—453,1972. K-BALB cells non-Inducible for type C viruses by 5-iododeoxy-uridine. Gen. Virol., 38: 419-429, 1978.

J.

25. Kaplan, J. C., Wilbert, S. M., Collins, J. J., Rakusanova, T., Zanonsky, G. B.. and Black, P. H. Isolation of simian virus 40-transformed induced hamster

6. Aoyagi, T., and Umezawa, H. Structures and activities of protease inhibitors of microbial origin. In: E. Reich, D. R. Rlfkln, and E. Shaw (eds.), Proteases and Biological Control, pp. 429-454. New York: Cold Spring Harbor, 197S.

cell lines heterogeneous for virus induction by chemicals or irradiation. Virology, 68: 200—214,1975.

7. Besmer, P.. Smotkln, D., Haseltine, W., Fan, H., Wilson, A., Paskind, M.,

Weinberg, R.. and Baltimore, D. Mechanism of Induction of RNA tumor viruses by halogenated pyrimidines. Cold Spring Harbor Symp. Quant. Biol. 39:1103-1007,1975. 8. Borek, E., and Ryan, T. Lysogenic induction. Prog. NucI. Acid Res. Mol. Biol., 249-300, 1973. 9. Cabradllla, C., Robbins, K., Aaronson, S. Induction of mouse type C virus by translational Inhibitors: evidence for a transcriptional derepression of a specific

class

of endogenous

virus.

Proc.

NatI. Acad.

Sd.

U.S.A.,

73: 4541 -

4545, 1976. 10. Capecchl, M. R., Capecchl, N. W., Hughes, S. H., and WahI, G. M. Selective degradation of abnormal proteins in mammalian tissue culture cells. Proc. NatI. Acad. Sd. U. S. A., 71: 4732—4736,1974. 11. Castellot, J. J., Miller, M. R., and Pardee, A. B. Animal cells reversibly permeable

to small

molecules.

Proc.

NatI. Acad.

Sci. U. S. A. 75: 3@1-3@@,

26. Lal, M. M., and Duesberg, P. H. Adenylic acid-rich sequences in RNA of

Rous sarcoma virus and Rauscher mouse leukemia virus. Nature (Lond.), 235:383-386,1972. 27. Long, C. W., Suk, W. A., and Greenawalt, C. Activation of endogenous type

C virus by amino acid alcohols. Virology 88: 194—1 96, 1978. 28. Lowy, D. R., Rowe, W. P., Teich, N., and Hartley, J. W. Murine leukemia

virus: high frequency activation in vitro by 5-lododeoxyuridine and 5-bro modeoxyurldine. Science, 174, 155—156,1971. 29. Manor, H., and Near, A. Effects of cycloheximide on virus DNA replication

in an Inducible line of polyoma-transformed rat cells. Cells, 5: 311—318, 1975.

30. Meyn, M. S., Rossman, T., and Troll, W. A protease inhibitor blocks SOS functions In Escherichia coil: antipain prevents A repressor inactivation, ultraviolet mutagenesis, and fllamentous growth. Proc. NatI. Acad. Sci. U. S. A.,74:1152—1156,1977. 31 . Penman, S. In: K. Abel and N. Salzman (eds.), Fundamental Techniques in

Virology, Chap. 5, pp. 35—48.New York: Academic Press, Inc., 1969.

1978.

12. Dunn, C. Y., Aaronson, S. A., and Stephenson, J. Interactions of chemical inducers and steroid enhancers of endogenous mouse type-C RNA viruses. Virology 66: 579-588, 197S. 13. Fogel, M. Induction of virus synthesis in polyoma transformed cells by DNA antimetabolites and by Irradiation after pretreatment with 5-bromodeoxyuri dine. Virology, 49: 12—22,1972. 14. Gerber, P. ActIvation of Epstein-Barr virus by 5-bromodeoxyuridine in virus free―human cells. Proc. NatI. Acad. Sci. U. S. A., 69: 83—85,1972. 15. GillespIe, D., Marshall, S., and Gallo, R. C. RNA of RNA tumor viruses contains poly A. Nat. New Biol. 236: 227—231 , 1972. 16. Goldberg, A. L., and Dice, J. F. Intracellular protein degradation in mam malian and bacterial cells. Annu. Rev. Biochem. 42: 835-869, 1974. 17. Goldberg, A. L., and St. John, A. C. Intracellular protein degradation in

mammalian and bacterial cells: Part 2. Annu. Rev. Biochem., 45: 747—803, 1976. 18. Goldschmldt, R. In vivo degradation of nonsense fragments In E. coil. Nature (Lond.)228: 1151—1154,1970. 19. Green, M., and Cartas, M. The genome of RNA tumor viruses contains poly A sequence. Proc. Natl. Acad. 3d. U. S. A., 69: 791-794, 1972. 20. Greenberger, J. S., and Aaronson, S. A. Cycloheximide Induction of xeno

tropic type C virus from synchronized mouse cells: metabolic requirements for virus activation. J. Virol., 15: 64—70,197S. 21 . Hamper, B., Derge, J. G., Martos, L. M., and Walker, J. C. Synthesis of

Epstein-Barr virus after activation of the viral genome In a negative human lymphoblastoid cell, Ragi, made resistant to 5-bromodeoxyurldine. Proc. NatI. Acad. Sci. U. S. A., 69: 78-82, 1972. 22. Hamper, B., Lenoir, G., Nonoyama, M., Derge, J. G., and Chang, S. Cell

cycle dependence for activation of Epstein-Barr by inhibitors of protein synthesis or medium deficient In arginine. virology, 69: 660—668,1976. 23. Johnson, L. F., Abelson, H. T., Green, H., and Penman, S. Changes in RNA

32. Plaft, T., Miller, J. H., and Weber, K. In vivo degradation of mutant Icc

repressor. Nature (Lond.); 228: 1154—1 156, 1970. 33. Radman, M. SOS repair hypothesis: phenomenology of an Inducible DNA

repair which Is accompanied by mutagenesis. In: P. C. Hanawalt and R. B. Setlow (eds.), Molecular Mechanisms for Repair of DNA, Basic Life Science 5cr. Vol. 5 pp. 355—367.New York: Plenum Publishing Corp., 1974. 34. Rascatl, R. J., and Tennant, R. W. Induction of endogenous murine retrovirus

by hydroxyurea and related compounds. Virology 87: 208—21 1, 1978. 35. Roberts, J. W., and Roberts, C. W. Proteolytic cleavage of bacteriophage

lambda repressor In Induction. Proc. NatI. Acad. Sd. U. S. A., 72: 147— 151, 1975. 36. Schlmke, R. T., and Bradley, M. 0. Properties of protein turnover in animal

cells and a possible role for turnover in “quality' ‘ control of proteins. In: E. Reich, D. B. Rlfkin, and E. Shaw(eds.), Protease and Biological Control, pp. 515-530. New York: Cold Spring Harbor, 1975. 37, Shlnagawa, H., and Itoh, T. Inactivation of DNA binding activity of repressor

in extracts of A-Iysogen treated with mitomycin C. Mol. Gen. Genet., 126: 103-110, 1973. 38. Singer, R. H., and Penman, 5. Messenger RNA In HeLa cells: kinetics of formation

and decay.

J. Mel.

Biol.,

78: 321 -334,

1973.

39. Smith, C. L, and Olshl, M. Early events and mechanisms In the induction of bacterial SOS functions: analysis of the phage repressor Inactivation process in vivo. Proc. NatI. Acad. Sci. U. S. A., 75: 1667-1661, 1978. 40. Telch, N., Lowy, D. R., Hartley, J. W., and Rowe, W. P. Studies on the mechanism of Induction of Infectious murine leukemia virus from AKR mouse embryo cell lines by 5-iododeoxyurldlne and 5-bromodeoxyuridlne. Virology,

51: 163—173, 1973. 41 . Tennant, R. W., Often, J. A., Quarles, J. M., Yang, W. K., and Brown, A. In:

J. M. Yuhas, R. W. Tennant, and J. D. Regan (eds.), Biology of Radiation Carcinogenesis, pp. 227—236.New York: Raven Press, 1976.

In relation to growth of the fibroblast. I. Amounts of mRNA, rRNA, and tRNA

AUGUST 1979

2999

Downloaded from cancerres.aacrjournals.org on July 9, 2011 Copyright © 1979 American Association for Cancer Research