Interactions of chemical inducers and steroid enhancers of endogenous mouse type-C RNA viruses

VIROLOGY 66, %%%8

(1975)

interactions

of Chemical Endogenous

CLAIRE

Y. DUNN,

STUART

Viral Carcinogenesis

Branch,

Inducers

Mouse

and Steroid

Type-C

A. AARONSON,

Enhancers

of

RNA Viruses AND

National

Cancer Institute,

Accepted

March

JOHN Bethesda,

R. STEPHENSON Maryland

20014

28, 1975

Biologically distinguishable endogenous type-C RNA viruses of BALB/c mouse cells are differentially affected by two classes of chemical inducers, halogenated pyrimidines and inhibitors of protein synthesis. In the present studies, the effects of these chemicals were compared on cells of genetic crosses involving BALB/c, C58, and NIH Swiss mouse strains. Cycloheximide was found to activate xenotropic vius from those genetic crosses from which xenotropic virus was inducible by IdU. In contrast, NIH Swiss-tropic endogenous viruses of BALB/c and C58 cells were much more resistant to activation by inhibitors of protein synthesis. Steroids possessing glucocorticoid activity enhanced virus release by cells exposed to either class of inducers. Unlike the inducers. which inhibited type-C virus release by exogenously infected cells, steroids augmented chronic virus production. These findings indicate that the mechanisms of action of inhibitors of protein synthesis and halogenated pyrimidines involve the virus-activation process, while steroids enhance rather than initiate virus synthesis. INTRODUCTION

The information for type-C RNA viruses is genetically transmitted within the highmolecular-weight DNA of normal mouse cells (Taylor et al., 1971; Stephenson and Aaronson, 197213; Rowe et al., 1972; Aaronson and Stephenson, 1973; Gelb et al., 1973; Chattopadhyay et al., 1974; Scolnick et al., 1974). Inbred mouse strains have been shown to contain at least three biologically distinguishable classes of endogenous viruses (Aaronson and Stephenson, 1973; Stephenson et al., 1974a; 1974c). Evidence has also accumulated that the number of endogenous viral genomes is strain dependent and that regulatory factors within the cell specifically affect the expression of each virus. Although the natural biologic functions of endogenous viruses are not well understood, it has been shown that at least one class of inducible type-C virus of the mouse is oncogenic (Stephenson et al., 1974b; Greenberger et al., 1975). Studies of the regulatory processes by which the cell controls type-C 579 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

virus expression may, thus, be of importance in establishing approaches to modify those endogenous viral functions detrimental to the host. In the investigation of intracellular regulatory processes, two highly potent chemical inducers of type-C virus have been utilized. These include halogenated pyrimidines (Lowy et al., 1971; Aaronson et al., 1971; Klement et al., 1971) and inhibitors of protein synthesis (Aaronson and Dunn, 1974b; Aaronson and Stephenson, 1975). both of which cause transient release of specific endogenous viruses from cells of the inbred BALB/c mouse strain. A third class of chemicals, glucocorticoids, has been reported to enhance type-C virus release from BALB/c cells in response to halogenated pyrimidines (Paran et al., 1973). In the present studies, we have investigated the effects of these chemicals on the activation of type-C virus from mouse embryo cells of well-characterized genetic crosses possessing biologically distinguishable endogenous type-C viruses.

580

DUNN, MATERIALS

AND

AARONSON

METHODS

Cells. Cells were grown in Dulbecco’s modified Eagle’s medium containing 10% calf serum (Colorado Serum Co., Denver CO) in 60 x 15-mm petri dishes (Falcon Plastics, Division B-D Laboratories, Inc., Los Angeles, CA). Continuous cell lines included NIW3T3 (Jainchill et al., 1969) and the normal rat kidney (NRK) line (Due-Nygen et al., 1955). The establishment of cell lines from individual NIH Swiss (NIH), BALB/c (BALB), NIHx(NIHxBALB)F,, (NIHxC58)F,, and (BALBxC58)F, embryos has been described in detail (Stephenson and Aaronson, 1972a; 1972b; 1973). Clonal lines of BALB and NIH cells, nonproductively transformed by the Kirsten strain of murine sarcoma virus (KiMSV), K-BALB, and K-NIH, have been previously described (Aaronson and Weaver, 1971). The isolation of KiMSV nonproducer clones derived from many individual NIHx (NIHxBALB)F, embryos and from (NIH x C58)F, and (BALBx C58)F, genotypes has also been reported (Stephenson et al., 1974a). NIHx(NIHxBALB)F, lines used included K-E8:2, K-ElO:l, and KE7:3. Viruses. The Rauscher (R) strain of murine leukemia virus (MuLV) and a clonal strain of KiMSV have been described (Aaronson and Weaver, 1971). Two prototype endogenous viruses of BALB cells, BALB: virus-l and BALB:virus-2, and an endogenous type-C virus of C58 cells, C58-MuLV, have also been reported (Stephenson and Aaronson, 1972a; Aaronson and Stephenson, 1973). Biologic assays. Virus induction was analyzed by a biologic assay utilizing cells nonproductively transformed by KiMSV. Chemical activation of these cells results in rescue of the sarcoma virus genome in the envelope of the endogenous type-C helper viruses of the cell (Aaronson, 1971; Klement et al., 1971). Whereas assays for helper leukemia viruses require multiple cycles of infection, transformation by MSV requires only a single cycle of infection (Aaronson and Rowe, 1970). Thus, this technique provides one of the most sensitive and quantitative biologic methods

AND STEPHENSON

available for studying induction of type-C viruses (Aaronson, 1971; Klement et al., 1971). The frequency of virus activation was measured by an infectious center assay as previously reported (Aaronson and Dunn, 1974a). Briefly, KiMSV nonproducer cells were treated with chemical inducers for designated times. Cultures were then washed twice with medium, exposed for 1 hr to 25 pg of mitomycin C per ml and transferred at lo-fold dilutions to petri dishes containing around lo5 NRK or NIH/3T3 cells plated a day earlier in medium supplemented with 2 pugof polybrene per ml (Toyoshima and Vogt, 1969). Sarcoma virus focus formation was scored 7-9 days later. Infection of N-tropic (Class I) viruses, such as BALB:virus-1 and C58MuLV, was assayed on NIH/3T3 cells, while xenotropic (Class II) viruses, such as BALB:virus-2, were detected on NRK cells (Aaronson and Stephenson, 1973; Stephenson et al., 1974a). The percentage of virusactivated cells was determined from the number of MSV infectious centers divided by the total cells as measured by cell count 24 hr after transfer. Induced focus-forming virus in tissue culture fluids was assayed on either NIH/3T3 or NRK cells as previously described (Aaronson and Weaver, 1971). Type-C

viral

DNA

polymerase.

assay.

Virion-associated reverse transcriptase activity in tissue culture fluids was measured in reaction mixtures containing in 0.05 ml: 0.05 M tris(hydroxymethyl)aminomethane (Tris)-HCl, pH 7.8; 0.06 M potassium chloride; 0.002 M dithiothreitol (DTT); 2 x lo-’ M managanese acetate; 0.02 A 260polyriboadenylicoligodeoxythymidylic acid (12-18); 2 x lo-’ M [3HjTTP (5000 cpm/pmole); and 0.1% (v/v) Triton X-100. After incubation at 37” for 60 min, DNA synthesis was measured as described previously (Aaronson et al., 1971). The standard error for replicate assays of a given virus stock was &15%. The specificity of the reaction for detection of murine leukemia virus (MuLV) was shown by inhibition of enzyme activity by antibody directed against MuLV reverse transcriptase (Aaronson et al., 1971). Chemicals. Chemicals included iododeoxyuridine (IdU), hydrocortisone, dexa-

CHEMICAL

INDUCERS

AND STEROID

581

ENHANCERS

methasone, d-aldosterone, pregenolone, and mitomycin C (Sigma, St. Louis, MO). Cycloheximide was kindly provided by the Drug Development Branch, National Cancer Institute.

1

RESULTS

Influence of hydrocortisone on the frequency of induction of BALB:virus-2 from BALBIc cells. Previous studies have shown

that protein-synthesis inhibitors and halogenated pyrimidines induce a xenotropic endogenous virus, designated BALB : virus-2, at high frequency from BALB cells (Aaronson and Stephenson, 1973; Aaronson and Dunn, 1974b). The availability of sensitive and specific methods for quantitating the frequency of BALB: virus-2 activation from BALB cells nonproductively transformed by MSV, made it possible to investigate the influence of steroids on the frequency of virus induction in response to two primary inducers. As shown in Fig. 1, the proportion of KBALB cells registering as BALB:virus-2activated increased as a function of IdU concentration. The maximum frequency of virus-induced cells was 10-l.’ at an IdU dose of 10 pg/ml. In contrast, the frequency of virus-induced cells in response to hydrocortisone alone was less than 10m5 (data not shown). Simultaneous exposure of K-BALB cells to IdU and hydrocortisone (10 pg/ml) caused no detectable increase in the frequency of virus-positive cells at any IdU concentration tested (Fig. 1). The frequency of BALB:virus-2-induced cells also rose as a function of increasing cycloheximide concentration (Fig. 2), confirming previous findings (Aaronson and Dunn, 197413).However, in contrast to the results obtained above for IdU-induction of BALB:virus-2, exposure of cycloheximideinduced cells to hydrocortisone led to a striking increase in the fraction of cells registering as BALB:virus-2 activated. In fact, at each cycloheximide concentration tested, simultaneous exposure to hydrocortisone produced approximately a lo-fold higher frequency of virus activation than that observed with cycloheximide alone. In this experiment, as many as lo-‘.’ of cells treated with both drugs registered as virus

0.1

1.0

IdU CONCENTRATION

10 (&/ml)

FIG. 1. Effect of hydrocortisone on virus induction from BALB cells in response to IdU. Exponentially growing cultures containing around 5 x lo5 K-BALB cells were exposed to varying concentrations of IdU in the presence (A) or absence (A) of 10 @g/ml hydrocortisone for 18 hr at 37”. The medium was changed twice to remove the drug and the cells were then treated with 25 &ml mitomycin C for 1 hr and transferred at lo-fold dilutions in duplicate to petri dishes containing around lo5 polybrene-pretreated NRK cells. Infectious centers of KiMSV-transformed foci were scored 7-9 days later as previously described (Aaronson and Dunn, 1974a). The frequency of virus induction was calculated from the number of infectious centers divided by the total cell count at 24 hr after transfer to an empty petri dish.

induced, and in some experiments this level reached as high as 10-“.4. The maximum frequency of BALB:virus-2 activation from K-BALB cells treated with both drugs was invariably many fold greater than that achieved with the optimal concentration of cycloheximide alone. Effect of h.vdrocortisone on the magnitude of virus production by spontaneousl> and chemically induced cells. In an at-

tempt to explain the differential influence of hydrocortisone on BALB:virus-2 induction by IdU and cycloheximide, studies were performed to determine the magnitude of virus release per induced cell in response to each chemical. The frequency of virus activation from K-BALB cells was tested by infectious center assay as described above, while supernatant fluids of identically treated cultures were tested at 24-hr intervals for the presence of type-C virus. As shown in Table 1, untreated K-BALB cells registered as BALB:virus-2

582

DUNN, AARONSON AND STEPHENSON

induced at a frequency of only 1.3 per lo6 cells. Similarly, tissue culture fluids contained a barely detectable level of spontaneously activated virus ( 1.5FFU/10s cells/24 hr). This reflects the very low frequency of spontaneous BALB:virus-2 release previously demonstrated to occur

CYCLOHEXIMIDE CONCENTRATION (rrghnl)

FIG. 2. Effect of hydrocortisone on virus induction

from BALB cells in response to cycloheximide. Exponential growing cultures containing around 5 x lo5 K-BALB cells were exposed to varying concentrations of cycloheximide in the presence (0) or absence (0) of 10 rg/ml hydrocortisone. The frequency of virusinduced cells was determined as described in the legend to Fig. 1.

with these cells (Aaronson and Dunn, 1974a). Hydrocortisone increased the frequency of virus-positive cells approximately 2-fold and the amount of spontaneously released virus around 3-fold (Table 1). Hydrocortisone caused little or no increase in the fraction of IdU-activated cells; however, cells simultaneously treated with both IdU and hydrocortisone exhibited a 2.5 to 5fold increase in total virus release. The magnitude of BALB:virus-2 release into tissue culture fluids after exposure to cycloheximide was 1.6 x 10” FFU/ lo6 cells, and was increased by lo- to 20-fold in the presence of hydrocortisone. This paralleled the lo-fold increase in frequency of cells registering as BALB:virus-2 positive by infectious center assay. Thus, while hydrocortisone enhanced spontaneous, as well as IdU or cycloheximideinduced virus release, it only caused a marked increase in the frequency of cycloheximide-activated cells. Since cycloheximide induced a relatively low level of virus per cell, it seems likely that hydrocortisone allowed many more cells to achieve a threshold of virus release required to register as infectious centers. Virus induction

TABLE

by inhibitors

of protein

1

EFFECT OF HYDROCORTISONEON SPONTANEOUSAND CHEMICAL INDUCTION OF BALB:VIRUS-2 FROM BALB MOUSE CELLS

Treatment

Spontaneous control +hydrocortisone IdU control thydrocortisone Cycloheximide control + hydrocortisone

Virus-induced cells/106 cells”

Maximum virus release/induced cell

Virus released/lOB/cells (FFU/24 hr)* Hours 24

48

72

1.3 x 100 2.8 x 10”

1.5 x 100 4.7 x 100

NT NT

NT NT

1.1 1.6

6.0 x lOa 6.1 x lo3

5.0 x 10’ 2.4 x lo2

4.0 x 10s 1.6 x 10’

1.6 x 10’ 4.4 x 10’

2.7 7.2

5.0 x 103 5.2 x 10’

1.6 x IO2 2.0 x 108



0.03 0.04

.

aExponentially growing cultures of K-BALB cells containing around 5 x 10’ cells, untreated or exposed to cycloheximide (10 &ml), or to IdU (30 &ml), for 18 hr at 37” in the presence or absence of 10 &ml hydrocortisone were tested for the frequency of virus induction on NRK cells as described in the legend to Fig. 1. bTissue culture fluids of cultures treated in parallel were assayed at 24-hr intervals for focus-forming virus on NRK cells as described in Methods. NT means not tested.

CHEMICAL

INDUCERS

AND STEROID

transformants after KiMSV infection (Stephenson et al., 1974a). One NIHx(NIH x BALB)F, line, K-E&2, treated with IdU registered as positive for BALB:virus-1 on NIH cells at a frequency of 10e3.’ but yielded no detectable NRK-tropic virus (BALB:virus-2). Neither virus was induced by cycloheximide even in combination with hydrocortisone. In contrast, line K-ElO: 1 was activated by IdU to release BALB: virus-2 at a frequency of lo- 3.5and to release this same virus at a frequency of 10-3.8 by cycloheximide. Virus induction was enhanced by more than 25-fold in cells simultaneously exposed to cycloheximide and hydrocortisone. A NIHx(NIH x BALB)F, backcross line, K-E7:3, that was noninducible for either virus by IdU, was also refractory to induction by cycloheximide in the presence or absence of hydrocortisone. The above results indicate that only those cells possessing information required for IdU induction of BALB: virus-2, were inducible by cycloheximide.

synthesis from genetic crosses of BALB and NIH strains. In addition to BALB:virus-2,

BALB mouse cells are known to contain an endogenous N-tropic virus, designated BALB:virus-1, which is also IdU inducible. In contrast, NIH embryo cells in culture are not IdU inducible for either virus (Stephenson and Aaronson, 1972a). As shown in Table 2, IdU activated BALB:virus-1 and BALB:virus-2 from K-BALB cells at similar frequencies, lo-“.’ and lo-‘-‘, respectively. Cycloheximide induced BALB: virus-2 at a frequency of 10-‘.3; in the presence of hydrocortisone this frequency was increased lo-fold. In contrast, there was no detectable induction of BALB: virus-l from K-BALB cells treated with cycloheximide even in the presence of hydrocortisone. Exposure of a KiMSV-transformed nonproducer clone of NIH embryo cells, K-NIH, did not result in virus activation under any condition tested (Table 2). A large number of individual NIH x (NIH xBALB)F 1 backcross generation embryo lines have been established. Approximately one-quarter of these lines are inducible by IdU to release, respectively, BALB:virus-1, BALB:virus-2, both, or neither viruses (Stephenson and Aaronson, 1972a; Aaronson and Stephenson, 1973). Clonal lines representative of each category have been derived as nonproducer

Cycloheximide induction of C58)F, and EALBxC58)F, hybrid

Genotype

2

INDUCTION OF BALB:VIRW1 NIH x (NIH ‘x BALB)F, BACKCROSS EMBRYO LINEP ON CYCLOHEXIMIDE

Clone designation

Log,, virus-induced IdU

BALB NIH NIHx(NIHxBALB)F,

K-BALB K-NIH K-E8:2 K-ElO:l K-E7:3

(NIH x embpo

cells. It is known that cells of the high leukemia incidence C58 strain contain genetic information for inducibility of multiple endogenous NIH-tropic viruses (Stephenson and Aaronson, 1973). Recent studies have indicated that these cells also contain infor-

TABLE EFFECT OF HYDROCORTISONE

583

ENHANCERS

cells/total

AND BALB:VIRW2

cells after treatment

Cycloheximide

NIH

NRK

-2.1 < -5.5 -3.5 < -5.5 c-5.5

-1.9 < -5.5 < -5.5 -3.5 < -5.5

NIH < < < < <

-5.5 -5.5 -5.5 -5.5 -5.5

NRK -2.3 < -5.5 < -5.5 - 3.8 < -5.5

FROM

with:

Cycloheximide thydrocortisone NIH <--5.5 < -5.5 < -5.5 < --5.5 c. 5.5

NRK 1.3 c -5.5 < -5.5 2.4 i -5..i

a Exponentially growing cultures containing around 5 x 10” cells of each KiMSV nonproducer clone were exposed either to IdU (30 &ml), cycloheximide (10 *g/ml), or cycloheximide (10 &ml) and hydrocortisone (10 pg/ml) for 18 h at 37”. The frequencies of induction of BALB:virus-1 and BALB:virus-2 were assayed on NIH/3T3 and NRK cells, respectively, as described in Methods. Spontaneous virus-activation frequencies either in the presence or absence of hydrocortisone, were less than 10mi 5 with each of the cell lines tested. The results represent the mean values of three separate experiments.

584

DUNN,AARONSONANDSTEPHENSON

mation for a xenotropic virus immunologically indistinguishable from BALB: virus-2 (Stephenson et al., 1974a). Each of these viruses has been isolated after induction by IdU of C58 parental cells and/or crosses containing genetic information of the C58 strain (Stephenson and Aaronson, 1972a; 1973). To determine the effect of inhibitors of protein synthesis on inducibility of these biologically distinguishable viruses of C58 origin, a KiMSVtransformed (NIH x C58)F 1 hybrid clone was treated with cycloheximide. Activation of a xenotropic (Class II) virus was observed at increasing frequency as a function of cycloheximide concentration; the maximum induction frequency of 10-3.5 occurred at a concentration of 100 pg/ml. Addition of hydrocortisone to treated cultures increased the frequency of virus-positive cells by around 30-fold (Fig. 3A). The same cells were also tested for induction of N-tropic (Class I) virus (Fig. 3B). High concentrations of cycloheximide

1 0.1

I 1.0

I 10

CVCLOHEXIMIOE

FIG. 3. Induction

I loo

T I

I 0.1

I 1.0

I 10

I loo

CONCENTRATION tug/ml)

of Class I and Class II viruses from (BALB x C58)F, hybrid cells. Exponentially growing cultures containing around 5 x 10’ cells of a KiMSV-transformed nonproducer (BALB x C58)F, hybrid clonal line were exposed to varying concentrations of cycloheximide in the presence or absence of 10 fig/ml hydrocortisone. The frequency of virus-induced cells was determined as described in the legend to Fig. 1. A. Virus-induction frequency in response to cycloheximide in the presence (0) or absence (0) of hydrocortisone assayed on NRK cells to test for Class II virus. B. Virus-induction frequency in response to cycloheximide in the presence (A) or absence (A) of hydrocortisone assayed on NIH/3T3 cells to test for Class I virus. Th arrows indicate virus-activation frequency below detection.

caused low but detectable virus activation. It can be seen that hydrocortisone increased by up to 50-fold the frequency of Class I virus-positive cells. These findings indicate that cycloheximide induces Class I as well as Class II virus from (NIH x C58)F, hybrid cells. Similarly, cycloheximide treatment of a (BALBxC58)F, hybrid embryo cell line resulted in activation of both classes of virus. In each case, the frequency of virus activation was markedly enhanced by steroid treatment (data not shown). The effects of other hormones on virus induction by cycloheximide. The influence

of other hormones on the frequency of virus induction in response to cycloheximide was next examined. As shown in Table 3, steroids including dexamethasone, hydrocortisone, and d-aldosterone were each highly potent enhancers of Class II endogenous virus release from a (BALBx C58)F, cell line. Under experimental conditions where cycloheximide caused virus induction at a frequency of 10-4.7, addition of dexamethasone increased the frequency to as high as 10-2.g. While hydrocortisone also markedly enhanced virus induction, a lOOfold greater concentration was required to achieve the same effect as dexamethasone. Methyl prednisolone and prednisolone (data not shown) were also potent enhancers of cycloheximide induction. In contrast, pregnenolone, another steroid, was inactive. Exposure of the same cell line to any of the steroids alone led to no detectable virus activation. Other hormones, including estradiol, testosterone, insulin, thyroxin, and epinephine, were inactive either as inducers or enhancers of cycloheximide activation of type-C virus (data not shown). Effect of hydrocortisone on chronic type-C virus release. The above studies

suggested that steroids increased the level of type-C virus release both spontaneously and after exposure to different classes of chemical activators. To test whether the actions of steroids were specific to the virus-induction process, the effect of steroid treatment was examined on chronic R-MuLV release by cultures of exogenously infected BALB mouse cells. As shown in

CHEMICAL TABLE

INDUCERS

AND STEROID

3

ENHANCEMENT OF CYCLOHEXIMIDE INDUCTION OF CLASS II VIRUS FROM (BALBxC%lF, CELLS BY HORMONES Hormone concentration b.tglml)

10-S lo..2 10 ’ 100 10' 102

Virus induction frequency in the presence of (log,, virus-induced cells/total cells) Dexamethasone

Hydrocortisone

d-Aldosterone

Pregnenolone

-4.7 -3.8 -2.9 NT NT NT

NT NT -4.3 -3.8 -3.7 -2.9

NT NT NT -3.8 -2.9 -2.9

NT NT NT -5.0 -4.9 -5.0

DExponentially growing cultures containing around 5 x lo5 KiMSV-transformed nonproducer (BALBxC58)F, cells were exposed to cycloheximide (10 &ml) for 18 hr at 37” in the presence of varying concentrations of dexamethasone, hydrocortisone, daldosterone, or pregnenolone. The frequency of induction of Class II virus was assayed on NRK cells as described in Methods. The frequency of virus activation in response to cycloheximide alone was 1O-‘-8. The results represent the mean of three separate experiments. NT means not tested.

585

ENHANCERS

tally distinct viruses segregated independently in appropriate genetic crosses (Aaronson and Stephenson, 1973). More recent evidence by molecular hybridization has indicated that there are differences in virus-specific sequences in the DNAs of prototype mouse strains (Chattopadhay et al; Scolnick et al., 1974) correspondmg to known differences in the number of loci for virus induction in these strains (Taylor et al, 1971; Stephenson and Aaronson, 197210. 1973, Rowe et al., 1972, Rowe, 1972). The above findings indicate that loci for virus induction represent viral structural information. It has previously been shown that halogenated pyrimidines and inhibitors 01 protein synthesis differentially affect two endogenous viruses of the BALB strain (Aaronson and Stephenson, 1973; Aaronson and Dunn, 1974al. While IdU induces both Class I and Class II viruses at high frequency, cycloheximide specifically induces Class II virus. The present studies TABLE

Table 4, hydrocortisone increased R-MuLV production by a factor of around 2.5-fold during the first 48 hr after treatment. In contrast, both IdU and cycloheximide were inhibitory to virus production by the same cells.

Treatment

DISCUSSION

The present studies have compared the effects of different chemical inducers on mouse embryo cells containing loci for induction of biologically distinguishable endogenous viruses. Evidence of spontaneous (Aaronson et al., 1969) and chemical (Lowy et al., 1971; Aaronson et al., 1971) activation of type-C RNA viruses from virus-negative mouse cells and findings that the high-molecular-weight DNA of mouse cells contained type-C viral nucleotide sequences (Gelb et al., 1972, 1973) demonstrated that these viruses were genetically transmitted. Other studies showed that virus inducibility was inherited as a dominant genetic characteristic (Stephenson and Aaronson, 1972a, Rowe, 19721 and that loci for induction of biologi-

4

EFFECT OF HYDROCORTISONE,IdU, AND CYCLOHEXIMIDE ON BALB CELLS CHRONICALLY INFEWFD WITH R-MuLV”

Control Hydrocortisone IdU Cycloheximide

Viral polymerase activity/ lo8 cells (pmoles TMP incorporated/ml Y lo-‘) at the following times after treatment 24 hr

48 hr

72 hr

20.0 55.0 12.0 1.5

18.0 49.0 s.0 8.0

20.0 25.0 1.o 1.5.0

-

a Exponentially growing cultures of R-MuLV-producing BALB cells, either untreated or exposed to hydrocortisone (10 &ml), cycloheximide (10 &ml), or IdU (30 &ml) for 18 hr at 37”, were medium changed twice to remove drugs. Tissue culture fluids were assayed at subsequent 24-hr intervals for virionassociated reverse transcriptase activity as described in Methods. Results are expressed as pmol [3H]TMP incorporated/lOe cells and represent mean values ot two separate experiments. The colony-forming efficiencies of parallel cultures measured 12 days after transfer at serial lo-fold dilutions to new petri dishes were as follows: untreated, 15”“/r; hydrocortisone. 12%‘: IdU, 2%; and cycloheximide, 5%.

586

DUNN,AARONSONANDSTEPHENSON

show that NIH embryo cells are not virus inducible by either chemical. Further, factors required for activation of Class II virus by cycloheximide were shown to be present in NIH x (NIH x BALB)F 1 backcross embryo cells that contained genetic factors necessary for induction of this virus by IdU. If loci for virus induction represent viral structural genes, the results argue that regulatory factors necessary for both cycloheximide and IdU induction of Class II virus are present in BALB and NIH cells or segregate in close approximation with the BALB locus for Class II viral structural information. Embryo cells derived from F, hybrids of the C58 strain and the noninducible NIH strain were found to be inducible by cycloheximide to release Class II virus. In addition, the same cells released Class I virus at a much lower frequency. It is known that while Class I viruses of C58 and BALB cells are similar in host range and immunologic properties, C58-MuLV is more infectious per physical particle than BALB:virus-1 (Stephenson and Aaronson, 1972a). Further, multiple loci for Class I virus have been demonstrated in C58 cells, while only a single locus for induction of this class of virus has been detected in BALB cells (Stephenson and Aaronson, 1972b; 1973). Whether the ability of cycloheximide to induce C58-MuLV but not BALB:virus-1 reflects quantitative differences in the biologic properties of these viruses or differences in the regulation of their expression by C58 and BALB cells remains to be determined. Recent studies have shown that induction of Class II virus by cycloheximide requires RNA synthesis and is associated with an increase in virus-specific RNA in treated cells (Aaronson et al., 19741.These findings have indicated that cycloheximide and other inhibitors of protein synthesis either impair cellular regulation of the transcription of viral RNA or its processing. While induction of type-C virus by IdU has been shown to require its incorporation into cellular DNA (Teich et al, 1973, Ilhe et al., 1974, Greenberger and Aaronson, 1975), the mechanism of induction is not yet known. As shown in the present stud-

,ies, both chemicals were potent activators of endogenous virus, even though they impaired chronic virus release by the same cells, exogenously infected with type-C virus. These results strongly imply that inhibitors of protein synthesis and halogenated pyrimidines act at steps specific to the virus activation process. In contrast, steroids were found to enhance release of type-C virus by infected cells as well as to augment induction of virus spontaneously and by chemicals. This suggests that steroids act to enhance rather than to initiate virus synthesis. Similar conclusions were recently drawn from studies of the effects of steroids on type-C virus release by AKR mouse cells (Ihle et al., 1975). Further evidence that steroids are not specific to the virus activation process comes from reports that these drugs also increase chronic production of other RNA-containing (Parks et al., 1974) as well as DNA-containing viruses (Morhenn et al., 1973). Steroids were found to markedly increase the frequency of virus-induced cells only in conjunction with cycloheximide. Cycloheximide caused activation of relatively small amounts of virus into tissue culture fluids. Thus, it seems likely that steroids simply cause a much higher fraction of cycloheximide-induced cells to achieve a threshold level of virus release necessary to register in the infectious center assay. In contrast, cells spontaneously activated or induced by IdU were sufficiently virus productive so that addition of steroids led to little, if any, increase in the frequency of virus induction. Those steroids that enhanced cycloheximide induction of virus all possessedglucocorticoid activity in the concentration range at which each was effective. Steroids have previously been shown to increase specifically the synthesis of certain cellular proteins (for review, see Lee and Kenney, 1971). The evidence indicates that their effects are mediated through a specific increase in transcription of the genes for these proteins (Kenney et ai., 1973; Rhoads et al., 1973; Harris et al., 1973). Recently, Wu et al. (1974) reported that these drugs affect type-C virus production posttranscriptionally since no in-

CHEMICAL

INDUCERS

AND STEROID

crease was detected in the level of virusspecific RNA in steroid-treated cells. Conversely, with mouse mammary tumor virus, steroid treatment was reported to be associated with increased B-type virusspecific RNA (Parks et al., 19’74). Thus, the mechanism of enhancement of virus synthesis by steroids also remains to be resolved. Further studies of the mechanisms involved in virus induction by chemicals specific to the activation process as well as how steroids enhance type-C virus production should aid in understanding cellular regulation of endogenous type-C viruses. ACKNOWLEDGMENT This work was supported in part by contract no. NCI-E-73-3212 of the Virus Cancer Program of the National Cancer Institute. REFERENCES AARONSON, S. A. (1971). Chemical induction of focus forming virus from nonproducer cells transformed by murine sarcoma virus. Proc. Nat. Acad. Sci.

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