EXPERIMENTAL
CELL
RESEARCH
19 1, 37-44
(1990)
Taxol Inhibits Stimulation of Cell DNA Synthesis by Human Cytomegalovirus REBECCA L. BALL,* DARRELL Departments
of ‘Microbiology
and PHuman
Biological
Chemistry
H. CARNEY,~ANDTHOMASALBRECHT**' and Genetics,
Inc.
INTRODUCTION Stimulation of cell DNA synthesis in quiescent cells appears to be a characteristic common to a number of transforming DNA viruses [l-5], and it has been suggested that stimulation of cell DNA synthesis is related to their oncogenic potential [6]. Human cytomegalovirus (CMV) infection of serum- or IUdR-arrested cells results in stimulation of cell DNA synthesis [5] and in an increase in the rate of cell division [7,8]. In addition, CMV infection has been associated with neoplastic disease [g-11], and it has been demonstrated that CMV malignantly transforms cultured hamster and human 1 To whom reprint Building, Route J-19, veston, TX 77550.
of Texas Medical
Branch,
Galveston,
Texas
77550
cells [12-141. Since stimulation of cell DNA synthesis may be related to the oncogenic potential of CMV and since a relationship between induction of a cellular proliferative signal and CMV replication has been proposed [15], it is important to determine the mechanism by which CMV initiates its mitogenic signal. A number of studies have suggested that altering the equilibrium between the microtubule (MT) polymer and unpolymerized tubulin can enhance or inhibit mitogenie signals. In some studies, colchicine, a MT-depolymerizing drug, blocks initiation of DNA synthesis [1621]. In other cases, increasing the level of unpolymerized tubulin subunits by colchicine treatment or microinjection ofphosphocellulose-purified tubulin subunits initiates DNA synthesis in serum-free fibroblast cultures [22; Carney, unpublished observations], or enhances stimulation of DNA synthesis in 3T3 cells treated with growth factors or hormones [23-261. Taxol, a drug which stabilizes intact MTs and stimulates polymerization of free tubulin, inhibits initiation of DNA synthesis in Go-arrested fibroblasts if added up to 8 h after thrombin or epidermal growth factor [27]. Together, these results suggest that a critical level of unpolymerized tubulin subunits may be required for cell progression through G,/G, and commitment to the S phase of the cell cycle. Alterations in the MT polymer could affect a number of early signal generating events in host cells including mobility of cell surface receptors or receptor interaction with signal generating molecules [see reference 28 for a review]. Other events which may occur later in the cell cycle, prior to entry into S phase, could also be affected. For example, a number of studies have shown that addition of growth factors to cells causes centriole duplication either at the Go/G, boundry or early in S [as]. In addition, the concentration of tubulin subunits has been shown to autoregulate the transcription and synthesis of tubulin [29-321. Thus, the extent of tubulin assembly plays a critical role in the regulation of new tubulin synthesis and, perhaps, in the synthesis of other molecules required for cell cycle progression. Experiments that have attempted to determine the temporal relationship between growth factor addition and MT-related events indicate that colchicine added
The microtubule (MT)-stabilizing drug, taxol, inhibited human cytomegalovirus (CMV)-initiated cell DNA synthesis by up to 100% in serum-arrested mouse embryo (ME) fibroblasts that were abortively infected by CMV. Taxol concentrations known to increase MT polymerization and to stabilize existing MTs (10 to 20 pg/ml) blocked CMV-stimulated cell DNA synthesis, while taxol concentrations of 2.5 pg/ml, or less, did not. Taxol maximally inhibited CMV initiation of cell DNA synthesis when added 3 h after virus infection and inhibited this initiation by greater than 50% when added up to 12 h after CMV infection. Control experiments suggest that taxol specifically inhibited CMV-stimulated cell DNA synthesis. Pretreatment of CMV stock with taxol did not reduce the stimulatory effect of CMV on cell DNA synthesis and taxol had no detectable effect on CMV-specific early protein synthesis. Moreover, taxol did not appear to alter thymidine pool sizes, affect cell viability, or compromise the DNA synthetic machinery in CMV-infected cells. Since taxol increases tubulin polymerization and inhibits MT disassembly, these results suggest that dynamic changes in MTs or in the pool of free tubulin subunits are necessary for CMV to stimulate cell entry into a proliferative cycle. (3 1990 Academic Press,
The University
requests should be addressed at Microbiology The University of Texas Medical Branch, Gal-
37
0014.4827/90
Copyright All rights
0
1990
of reproduction
by Academic in any form
$3.00
Press, Inc. reserved.
38
BALL,
CARNEY,
up to 10 h after growth factors can still enhance mitogenesis [23-261. In addition, in lymphocytes, where colchicine inhibits lectin-induced mitogenesis, this response is observed when colchicine is added up to 10 to 16 h after mitogen addition [ 16-171. These observations are consistent with our previous results showing that taxol can block initiation of DNA synthesis when added to cells up to 8 h after thrombin or epidermal growth factor [27]. In the present study, we have used taxol to examine if CMV initiates cell DNA synthesis by a mechanism that involves the equilibrium between free tubulin and the MT polymer. The present findings demonstrate that taxol blocks initiation of cell DNA synthesis by CMV in quiescent mouse embryo (ME) fibroblasts. These results suggest that the extent of MT polymerization or changes in the pool of unpolymerized tubulin subunits may be involved in regulating mitogenic signaling by CMV. Since previous studies have shown that other mitogenic signals involve dynamic changes in the MT-tubulin equilibrium, these studies suggest that such a MT-related signal may represent a common mechanism in growth factor and oncogenic virus stimulation of cell proliferation.
MATERIALS
AND
METHODS
Cell culture. Primary cultures of ME fibroblastic cells were obtained by trypsinization of the body wall of 9 to 13 day-old embryos as previously described [33]. ME cells were grown in 45% DulbeccoVogt’s modified Eagle’s medium, 45% Ham’s Fl2 medium (DV-F12 medium), 10% calf serum (growth medium), 100 U/ml penicillin, and 100 pg/ml streptomycin in a humidified atmosphere of 5% CO, in air at 37°C. Dulbecco-Vogt’s modified Eagle’s medium, Ham’s F12 medium, trypsin solution, glutamine, and antibiotics were obtained from Gibco (Grand Island, New York), and calf serum was purchased from Irvine Scientific (Irvine, CA). The derivation, culture, and maintenance of human embryonic thyroid (THY) and lung (LU) cells has been described in detail [34]. THY or LU cells were grown in 32 oz. glass prescription bottles in Eagle’s minimum essential medium (EMEM) supplemented with 10% y-irradiated fetal calf serum (yFCS) and 0.075% NaHCO,. Cell growth arrest. Quiescent cultures of ME cells were obtained by initially plating the cells at a density of 5 X lo* cells/cm* followed by incubation for 48 h in growth medium. The growth medium was then removed and replaced with medium without serum and incubation was continued for 10 min. The medium was changed to fresh serum-free medium and the cells were incubated at 37°C for 24 h. To provide a more synchronous population of cells, the cells were returned to growth medium for 2 h. Afterwards, the medium was removed, changed to serum-free medium as above, and the incubation in serum-free medium was continued for 48 h prior to virus infection. Virus propagation, infectivity assay, and control cell lysates. Strain AD169 of CMV was used for these studies and was propagated as described previously in detail [35]. When more than 90% of the cells demonstrated large nuclear inclusions for 3 days, the virus was harvested. Cells were scraped from the surface of the glass prescription bottles and the cells and debris were separated from the fluids by centrifugation at 184g. The clarified culture fluids were reserved. The pellet was resuspended in 5 ml of the reserved fluids and sonicated to lyse the cells. Debris was removed from the sonicated suspension by
AND
ALBRECHT
centrifugation as before and the clarified fluids were mixed with the reserved culture fluids (virus stock). Virus stocks were dispensed in 5 ml vials and stored at -70°C. Infectivities of the virus stocks were determined by plaque assay as previously described [34] and were in the range of 1 X lo6 to 2 X lo7 plaque forming units (PFU)/ml. Control mock-infected cell lysates were prepared from uninfected THY or LU cells in the same manner as virus stocks. CMV and mock infection. Serum-arrested ME cells in 35.mm dishes were infected with CMV as follows. The culture fluids were aspirated and the cells were inoculated with 0.5 ml of virus stock diluted in serum-free EMEM containing 0.075% NaHCO, to obtain a predetermined calculated multiplicity of infection. Control cultures were mock-infected with the same volume and dilution of cell lysate. The cells were incubated for 1 h at 37°C in 5% CO, with gentle rocking every 15 min to assure that the cells were constantly in contact with the inoculum. The inoculum was then aspirated and the cells were washed with serum-free DV-F12 and further incubated in fresh serum-free medium as described above (0 h postinfection, PI). Ultraviolet WV) irradiation. Virus stocks were UV-irradiated in 1 to 1.5 ml volumes with continuous stirring of the fluids. A GE-G8T5 bulb was used at a dose rate of 80 ergs/s/mm* as determined with a Blak-ray ultraviolet meter (Ultraviolet Products, Inc., San Gabriel, CA). Measurement of cell DNA synthesis. The effect of taxol on the mitogenic signal induced by CMV infection or mock infection was determined by measuring the rate of incorporation of [methyl-3H]thymidine ([3H]TdR, 20 Ci/mmol, ICN Radiochemicals, Irvine, CA) or [methyl-3H]hypoxanthine (10 Ci/mmol, New England Nuclear, Boston, MA) into cell DNA. [3H]TdR (1 &i/ml) was added at selected times after virus infection. At the end of each [3H]TdR pulse period, the cells were enzymatically digested and the DNA was released by the addition of 0.1% Protease XIV in 0.1X SSC (0.015 M NaCl, 0.0015 M Na citrate; pH 7.0), 2% sodium lauryl sarcosinate in 0.1X SSC, and 2% ethylenediaminetetraacetic acid (EDTA), and incubation at 37°C for 18 to 24 h [36]. Each sample was then titurated vigorously to shear the DNA, mixed thoroughly, and a 50 ~1 aliquot was applied to a 2.3 cm Whatman No. 3 filter paper disc to measure total DNA synthesis. DNA was precipitated onto the filter and filter papers were washed three times for 15 min each with cold 5% trichloroacetic acid (TCA). The filter papers were dried under a heat lamp or washed twice with ethanol and once with acetone, and air-dried overnight. Radioactivity was determined using 10 ml of 0.4% Omnifluor (New England Nuclear) in toluene. For labeling with [3H]hypoxanthine, an isotope concentration of 5 wCi/ml was used. Samples were treated after DNA extraction with 0.3 M KOH at 37°C for 12 h to hydrolyze labeled RNA before measurement of [3H]hypoxanthine incorporation into DNA as above [7]. Separation of uirus and cell DNA. The method of Crouch and Rapp [36] was used to separate virus and cell DNA in isopycnic gradients of CsCl. The DNA was centrifuged to equilibrium in CsCl gradients. The gradients were collected from the bottom of each tube in 8-drop fractions onto Whatman No. 3 filter paper discs. The radioactivity in each fraction incorporated into acid-precipitable material was determined as above. Samples were removed at regular intervals and the density was determined with an Abbe refractometer (Model B, Carl Zeiss, New York, NY). Autoradiography of labeled nuclei. The percentage of nuclei in which DNA was synthesized was determined by adding [3H]TdR (1 &i/ml) to CMVor mock-infected cells 24 h postinfection (PI). After a 6 h pulse, the cells were washed twice with PBS, fixed with 3% formaldehyde for 15 min at 4”C, washed with PBS, and extracted with cold 5% TCA for 15 min. The fixed cells were washed 3 times with PBS and allowed to air-dry. A thin film of Ilford K5 autoradiographic emulsion was applied to each dish and exposed for 2 weeks at ~20°C. The emulsion was developed and the nuclei stained with 2 ml of Hoeschst stain (5 wg/ml in PBS) for 30 min at 23°C. Nuclei that
CMV-INDUCED
CELL
DNA
-
incorporate [“HJTdR are covered with autoradiographic grains and all nuclei are fluorescent. Each culture was examined with a Leitz microscope with phase conkast and epifluorescence illumination. A minimum of 1500 labeled and unlabeled nuclei were scored for each determination. CelE ~~~bil~ty assay. At selected times PI, ME cells were removed from 35-mm dishes with trypsin solution. The cells were pelleted at 184g for 20 min. The supernatant fluids were decanted and the cell pellet was gently resuspended in trypan blue (10% in PBS). The percentage of viable (dye-excluding) cells in each sample was determined with the aid a hemocytometer. Immunofluorescence assay for CMV antigens. ME cells, grown on glass coverslips in Xi-mm dishes (2 X 105 cells/dish) were serumarrested and infected as described above. At selected times PI, the inoculum was aspirated and the cells were washed three times in PBS and fixed for 10 min in acetone (-20°C). Following fixation, the cetls were washed in PBS and incubated for 30 min at 37°C with human convalescent serum reactive to CMV strain AD169. The cells were then washed in PBS and incubated for 30 min with a fluorescein isothiocyanate-conjugated goat anti-human IgG. The cells were washed again in PBS and t,he coverslips were mounted on glass slides in PBS:glycerol (1:l). The cells were examined with a Zeiss fluorescence microscope. Drugs. Taxol was supplied by Dr. Matthew Suffness, National Products Branch, National Cancer Institute. Tax01 was disolved in ethanol to obtain a stock solution of 1.0 mg/ml and diluted with serum-free DV-F12 medium to achieve a final concentration of 10 @g/ml, unless indicated otherwise.
RESULTS
Absence
of HCMV
DNA
Synthesis
in ME Cells
Boldogh et al. [37] have reported that ME cells sustain an abortive infection with CMV in which CMV DNA synthesis is undetectable. Since the absence of CMV DNA synthesis in ME cells would facilitate this study by eliminating the need to separate cell and CMV DNA, we first attempted to confirm the findings of Boldogh and his colleagues. [3H]TdR-labeled DNA from CMV-infected ME cells and 14C-labeled DNA from mock-infected cells were ~osedimented to their buoyant densities in cesium chloride gradients. Consistent with the results of Boldogh et al. [37], synthesis of CMV DNA was not detected through 144 h PI in serum-arrested ME cells (data not shown). Furthermore, as previously reported by these investigators i37J, infectious yields of progeny CMV were not obtained from ME cells (data not shown). Thus, since CMV infection of ME cells (like infection of bovine, guinea pig, and hamster cells [7,38, 391) does not result in detectable synthesis of CMV DNA, the t3HJTdR incorporation observed in experiments with ME cells reflects cell DNA synthesis. Effect of Tax01 on Initiation by CMV
of Cell DNA Synthesis
To determine if the MT-tubulin equilibrium is involved in mitogenic signaling by CMV, serum-arrested ME cells were infected with CMV (1 PFUlcell) or were mock-infected with uninfected-cell lysate, and were
39
SYNTHESIS /
/
I
I
24
30
1
I
/
36
42
48
. CMV 0 CMV + taxoi A Lysate
A Lymte + tax01
LL..--~&.---li_..~ 0 6
12
18 Time
Post-lnfectlon
(h)
FIG. 1. Effect of taxol on CMV-initiated cell DNA synthesis. Serum-arrested ME cells were infected with 1 PFU/cell of CMV (circles) or were mock-infected (triangles) with the same amount and dilution of a cell lysate. Taxol(l0 pg/ml) was added at 3 h PI and was present for the duration of the experiment (open symbols). 13H]TdR (1 pCi/ml) was added for consecutive 6 h pulses through 48 h PI. Incorporation of 13H]TdR into acid-precipitable material was measured at the end of each pulse as described under Materials and Methods. Each dat,um point is plotted at the midpoint of the pulse period and represents the means of triplicate determinations.
treated with taxol (10 gg/ml) for the duration of the experiment or were left untreated. ~3H]TdR incorporation was measured for consecutive 6 h pulses through 48 h PI. As shown in Fig. 1, maximum stimulation of cell DNA synthesis by CMV was observed 24 to 30 h PI and was 2.5-fold greater than in control cultures. This stimulation was due to the presence of CMV since cells infected with UV-irradiated CMV stock (2.88 X lo5 ergs/mm’), which eliminated CMV infectivity, demonstrated levels of [3HJTdR incorporation comparable to those observed in cells mock-infected with uv-irradiated or with nonirradiated cell lysate (1.1 + 0.032 X 104 CPM for cells infected with irradiated CMV compared to 1.4 f 0.22 X lo4 or 1.4 ?z 0.043 X lo* for cells treated with nonirradiated or irradiated cell lysates, respectively). Taxol inhibited CMV-initiated cell DNA synthesis during this pulse period by about 75% suggesting that a pool of free tubulin subunits might be necessary for CMV to initiate a mitogenic signal. Influence of Taxol Concentration DNA Synthesis
on CMV-Initiated
Cell
The concentration dependence for tax01 inhibition of CMV-initiated cell DNA synthesis was determined by treating CMV- or mock-infected cultures with 0.3 to 20 pg/ml of taxol for the duration of the experiment and measuring 13H]TdR incorporation during a 6 h pulse from 24 to 30 h PI. As shown in Fig. 2, taxol concentrations of 2.5 pg/ml, or less, inhibited CMV-initiated cell DNA synthesis by less than 20%. In contrast, 10 or 20 pug/ml of taxol caused 100% inhibition of CMV-initiated
40
BALL,
CARNEY,
60 40 20
Taxol(pg/ml)
FIG. 2. The concentration effect of taxol on CMV-initiated cell DNA synthesis. The procedure was as described in the legend to Fig. 1 except that taxol was added at the concentrations indicated and [3H]TdR incorporation was determined 24 to 30 h PI. The data are derived from a representative experiment and are plotted as percent inhibition of CMV-stimulated [3H]TdR incorporation. The variation in the triplicate determinations used to calculate each datum point did not exceed 10%.
[3H]TdR incorporation, and half-maximal inhibition of CMV-induced cell DNA synthesis was observed at a taxol concentration of 6 pg/ml. These concentrations correspond to previously reported taxol concentrations required to prevent MT depolymerization in ME cells by colchicine [40, 411. Thus, these results suggest that taxol inhibition of CMV-stimulated DNA synthesis may be a direct result of taxol-induced alterations in the MT-tubulin equilibrium. The Time Course of Taxol Inhibition Cell DNA Synthesis
of CMV-Initiated
Because previous studies suggested that altered MTs might affect early events in cell entry into S phase, we investigated the time course for taxol sensitivity in CMV-infected ME cells. Taxol (10 pg/ml) was added at selected times after CMV infection (1 PFU/cell) and [3H]TdR incorporation was measured 24 to 30 h PI. Taxol added up to 9 h PI inhibited 70 to 100% of CMVstimulated cell DNA synthesis (Table 1). When added at 12 h PI, taxol caused only 50% inhibition suggesting that the taxol-sensitive event(s) had taken place in most cells by this time. Since taxol had little effect when added by 18 to 24 h PI, it appears that taxol did not nonspecifically impair the DNA synthetic machinery. In addition, at each of the time points shown, taxol caused an actual inhibition of CMV-initiated [3H]TdR incorporation and did not simply delay the onset of DNA synthesis (Fig. 1 and data not shown). Absence of a Direct Tax01 Effect on CMV To investigate the possibility that taxol might inactivate CMV, CMV stock was pretreated with taxol (10 pg/ml) at 37°C for 1 h. The virus stock was then diluted
AND
ALBRECHT
SO that a final calculated multiplicity of infection of 1 PFU/cell and a final taxol concentration of 0.5 pg/ml were achieved. As shown in Table 2, there was no significant difference in stimulation of [3H]TdR incorporation in serum-arrested ME cells infected with untreated CMV stock or with CMV stock pretreated with taxol. In the same experiment, however, 100% inhibition of CMV-initiated [3H]TdR incorporation was observed when 10 pg/ml of taxol was added 3 h PI. Accordingly, taxol did not exert its inhibitory effect on CMV-induced cell DNA synthesis by acting directly on the virus. To determine if taxol nonspecifically inhibited CMV early protein synthesis in ME cells, CMV-infected ME cells were treated with 10 pg/ml of taxol or were left untreated, and were examined by indirect immunofluorescence using human convalescent serum highly reactive to CMV antigens. No difference was noted in the percent of cells demonstrating immunofluorescence, in the location, or in the intensity of the staining of taxoltreated or untreated CMV-infected cells (data not shown), suggesting that taxol does not inhibit CMV early protein synthesis. These results also exclude the possibility that taxol had a substantial effect on adsorp-
TABLE Time
Time of tax01 addition relative to infection M 0
Course
of Taxol [3H]Thymidine
Viral-initiated [3H]thymidine incorporation (CPM t SD x IO-“)”
3 6 9 12
10.8 8.8 8.4 11.2 12.4
f k f + f
1.2 1.3 1.2 1.2 0.95
18
12.3 ? 1.3
24
11.7
4 0.51
1
Inhibition of CMV-Initiated Incorporation Viral-initiated [“Hlthymidine incorporation in the presence of taxol* (CPM + SD x lo-“) 4.4 k 0.82 0 1.4 2 0.19 3.3 t 0.53 5.8 * 0.35 9.9 T!c0.85 9.6 k 1.2
Inhibition (%)’ 60 100 83 70 53
19 18
o Values (CPM: counts per minute) represent the means of triplicate determinations from one representative experiment and are the difference in radioactivity for CMVand mock-infected cells in the absence of taxol treatment. The standard error of the difference = \/(SD,)‘/3 + @D&‘/3, w h ere SD, = standard deviation of the mean radioactivity for CMV-infected cells and SD, = standard deviation of the mean radioactivity for mock-infected cells. (1 Taxol (10 pg/ml) was added at the times indicated relative to removal of the inoculum (0 h) and was present for the duration of the experiment. [3H]thymidine (1 &i/ml) incorporation was determined 24 to 30 h PI. Values are the difference in radioactivity for taxoltreated, CMVand mock-infected cells. The standard error of the difference was calculated as described in footnote a. ’ Percent inhibition = [(V - L) - (V, ~ LT)] /V - L X 100, where V = radioactivity for CMV-infected cells, L = radioactivity for mock-infected cells, VT = radioactivity for CMV-infected cells treated with taxol, and L, = radioactivity for mock-infected cells treated with taxol.
CMV-INDUCED TABLE
TABLE
2
Effect of Pretreatment of CMV with Taxol on Virus Initiation of Cell DNA Synthesis Treatment CMV (1 PFU/cell) CMV pretreatment with taxol’ CMV with taxol’ added 3 h PI Cell lysate Cell lysate + taxol’
(CPM
Radioactivity f SEM X 10m4)”
3
Effect of Taxol on CMV Initiation of Cell DNA Synthesis as Determined by Measuring [3H]Hypoxanthine Incorporation
Stimulation of cell DNA synthesis (%jb
2.6 k 0.23
52
2.3 tr 0.14d
45
1.4 * 0.4Bd 1.7 -t 0.11 1.6 f 0.051
41
CELL DNA SYNTHESIS
0
’ Incorporation of [3H]thymidine (1 &i/ml) was determined 24 to 30 h PI. Values (CPM: counts per minute) represent the means of triplicate determinations from one representative experiment i standard error of the mean (SEM). b Percent stimulation of cell DNA synthesis = (V - L)/L X 100, where V = radioactivity for CMV-infected cells and L = radioactivity for mock-infected cells. ’ At 10 pg/ml, added to virus stock at 37°C 1 h prior to infection of ME cells. d Comparison of the data for untreated CMV and CMV pretreated with taxol by the Knuskal-Wallis and multiple comparison tests indicated that these numbers were not significantly different with an overall significance level of (Y = 0.05. Statistical analysis of the dat,a for cells infected with either untreated CMV or CMV pretreated with taxol indicated that these numbers were significantly different from the data for CMV-infected cells treated with taxol at 3 h PI or cells mock-infected with cell lysate. ‘At 10 pg/ml, added 3 h PI and present for the duration of the experiment.
tion or penetration of CMV. Therefore, taxol does not appear to affect CMV virions or synthesis of CMV early proteins. Evidence that Tax01 Effects on CMV-Stimulated DNA Synthesis Are Specific for Mitogenic Signal Events Studies of the specificity of taxol in ME cells have shown that taxol does not affect transport of small molecules, protein or RNA synthesis, cell morphology, or cell viability [27]. To exclude the possibility that taxol might nonspecifically affect CMV-infected ME cells, the following experiments were undertaken. First, to ensure that taxol caused a true inhibition of CMV-initiated [3H]TdR incorporation rather than inducing a change in thymidine pool sizes in CMV-infected ME cells, [3H]hypoxanthine was used to measure DNA synthesis. Taxol(l0 pglml) inhibited CMV-initiated cell DNA synthesis by 72% as measured by [3H]hypoxanthine incorporation (Table 3). Since the level of this inhibition is similar to the level of inhibition observed when [3H]TdR was used to measure DNA synthesis, taxol inhibition of CMV-induced cell DNA synthesis does not appear to be a consequence of a change in TdR pool sizes.
Radioactivity (CPM + SEM x 10m4)”
Treatment CMV CMV + taxold Cell lysate Cell lysate + taxold
12.3 9.4 7.7 8.1
k f zk f
0.12 1.0 0.38 0.24
Stimulation of cell DNA synthesis (%Y 62 16
Inhibition by taxol (%Y
72
’ Incorporation of [3H]hypoxanthine (5 pCi/ml) was determined 24 to 30 h PI. Values (CPM: counts per minute) represent the means of triplicate determinations from one representative experiment * standard error of the mean (SEM). * Percent stimulation of cell DNA synthesis = (V - L)/L X 100, where V = radioactivity for CMV-infected cells and L = radioactivity for mock-infected cells. ’ Percent inhibition = [(V - L) - (V, - L,)]/V - L X 100 where V = radioactivity for CMV-infected cells, L = radioactivity for mock-infected cells, V, = radioactivity for CMV-infected cells treated with taxol, and L, = radioactivity for mock-infected cells treated with taxol. dAt 10 pg/ml, added 3 h PI and present for the duration of the experiment.
Second, trypan blue exclusion was used to determine if cell death might account for the inhibition of CMV-initiated cell DNA synthesis by taxol. Cells were examined at 24 and 30 h PI for the ability to exclude dye (Table 4). As shown, taxol did not affect the viability of CMV-infected ME cells. Third, light microscopic examination showed no change in morphology of CMV-infected ME cells associated with taxol treatment. Fourth, we determined if taxol might inhibit the rate or extent of DNA synthesis in CMV-infected ME cells rather than the entry of cells into S phase. This distinction can be made by determining the actual number of cells synthesizing DNA. As shown in Table 5, the percentage of labeled
TABLE
4
Effect of Taxol on Cell Viability in CMV-Infected ME Cells Determined by Trypan Blue Exclusion Treatment CMV CMV + taxol* Cell lysate Cell lysate + taxol*
Viable cells (%)” 96.4 96.8 94.2 95.4
a Percent viable cells = dye-excluding cells/dye-excluding + stained cells X 100. A minimum of 250 cells were counted datum point. bAt 10 pg/ml, added 3 h PI and present for the duration experiment.
cells for each of the
42
BALL,
TABLE Effect of CMV-Infected Autoradiography
Taxol
on Mouse
Incorporation Embryo
CARNEY,
ANL)
5 of Cells
[“HjThymidine as Determined
in by
Labeled nuclei (%Y
Treatment CMV (1 PFU/cell) CMV + taxol’ Cell lysate Cell lysate + taxol’
21 9 8 6
o The percentage of labeled radiographically labeled and minimum of 1500 cells from b At 10 pg/ml, added 3 h experiment.
-
nuclei was determined by counting autononlabeled Hoechst-stained nuclei in a random fields. PI and present for the duration of the
nuclei determined by autoradiography was 2.5-fold greater in CMV-infected cultures than in control cultures. This increase correlates with the level of CMVstimulated [3H]TdR incorporation in ME cells. Thus, the CMV-induced stimulation of cell DNA synthesis represents an increase in the number of cells entering a proliferative cycle. Taxol (10 yglml) inhibited CMV-induced stimulation of nuclei labeling by nearly lOO%, confirming that taxol inhibits the initiation of cell DNA synthesis by CMV rather than the rate or extent of DNA synthesis.
DISCUSSION
The data presented in this report suggest that CMV, an oncogenic DNA virus, initiates cell DNA synthesis by a mechanism involving an alteration in the MT-tubulin equilibrium. Taxol inhibited CMV-initiated [3H] TdR incorporation by up to 100% suggesting that a critical concentration of free tubulin subunits, or changes in the extent of polymerization of existing MTs are required for mitogenic signaling by CMV. The concentration dependence of the taxol inhibition of CMV-initiated DNA synthesis parallels the concentration dependence for taxol stabilization of MTs and taxol inhibition of DNA synthesis stimulated by growth factors. Taxol concentrations of 2.5 pg/ml, or less, did not substantially inhibit initiation of t3H]TdR incorporation by CMV, while 10 or 20 pug/ml of taxol blocked CMV-initiated t3H]TdR incorporation. Previous studies in which MTs were visualized by antitubulin immunofluorescence [40] or experiments in which MT content was quantified by binding of a radioactive monoclonal antibody to tubulin [41] have shown that colchicine (1 PLM) depolymerizes most MTs in cells treated with
ALBRECHT
less than 5 Kg/ml of taxol. In contrast, 5 and 10 to 20 pug/ml of taxol partially and totally stabilize MTs against disruption by 1 pM colchicine, respectively. The taxol concentration dependence for inhibition of CMVinitiated cell DNA synthesis is similar also to the taxol concentration dependence for inhibition of initiation of DNA synthesis by growth factors. For example, taxol concentrations of less than 5 Fg/ml do not substantially inhibit thrombin-initiated DNA synthesis, while 5 wg/ ml and 10 to 20 pglml cause half-maximal and maximal inhibition, respectively [27]. Thus, the similarities in the concentration dependence for taxol inhibition of CMV- and growth factor-initiated DNA synthesis and inhibition of MT disruption by 1 yM colchicine suggest that these mitogenic agents share a common mechanism for initiation of cell DNA synthesis involving the MT-tubulin equilibrium. The time course for taxol inhibition of DNA synthesis also suggests that taxol inhibits an event(s) common to both growth factor and CMV initiation of cell proliferation. Taxol inhibited CMV initiation of cell DNA synthesis by 70 to 100% when added to quiescent ME cells up to 9 h after CMV infection. By 12 h PI taxol inhibited approximately 50% of the CMV-induced stimulation of cell DNA synthesis, and after this time had little effect. Previous studies have shown that taxol inhibits thrombin- or epidermal growth factor-initiated DNA synthesis in ME fibroblasts maximally when added during the first 6 hrs after the growth factor and is 50% as effective by 8 h [27]. Thus, taxol inhibits initiation events for a somewhat longer period of time after CMV infection than after growth factor treatment. The maximum rate of TdR incorporation after growth factor treatment of ME cells is from 20 to 24 h compared to 24 to 30 h for CMV stimulated cells. Accordingly, the time course of taxol sensitivity of CMV-stimulated cell DNA synthesis may be extended somewhat due to the requirement for synthesis of early CMV or cellular proteins which may be involved in mitogenic signaling [13,15]. The observation that taxol interrupts the proliferative signal generated by CMV and growth factors with similar timing suggests that both of these mitogenic agents may require changes in the MT-tubulin equilibrium during a critical period to commit cells to DNA synthesis. Although taxol appears to specifically block CMV initiation of t3H]TdR incorporation in a manner similar to its inhibition of growth factor-induced mitogenesis, it is not clear if this inhibition is due to a decrease in the pool of free tubulin and/or stabilization of preexisting MTs. Alternatively, taxol may restrict specific MT-directed events which might require the regulated polymerization of new tubulin subunits such as reorganization of the golgi apparatus and the splitting and maturation of centrioles. Interestingly, both of these events occur late
CMV-INDUCED
CELL
in G, prior to cell entry into S phase and correlate with the ability of cells to enter S phase [28, 421. A number of studies have demonstrated that MTs are disrupted or disorganized in transformed cells [43-481, leading to the hypothesis that a sustained increase in the level of unpolymerized tubulin may be related to uncontrolled proliferation in transformed cells. Pfeiffer et al. have shown that CMV infection results in MT depolymerization in some fibroblastic cells [49]. In our studies, although taxol-stabilized MTs appeared similar in CMV- and mock-infected cells, it was not possible to demonstrate a correlation between CMV stimulation of cell DNA synthesis and depolymerization of MTs (data not shown). To observe individual MTs by immunofluorescence with an acceptable level of background staining, low cell densities were required where neither growth factors nor CMV stimulate cell DNA synthesis effectively. Nevertheless, the lack of observable MT depolymerization in CMV-infected cells, when considered together with our findings for the taxol effect on CMVinitiated cell DNA synthesis, suggests that CMV-induced mitogenesis may require a critical free tubulin pool size or changes in functional subsets of MTs [50], rather than affecting gross changes in the total cytoplasmic MT complex. Recent evidence suggests that unpolymerized tubulin dimers may be directly involved in modulation of tubulin mRNA levels in cells. Ben-Ze’ev and Cleveland showed that tubulin autoregulates its synthesis indicating that the free tubulin pool size controls tubulin synthesis [29,30]. For example, tubulin synthesis is rapidly and specifically depressed in cells treated with drugs such as colchicine that increase free tubulin content [30] or in cells microinjected with exogenous tubulin [31]. In cont,rast, vinblastine, which decreases the free tubulin pool size by forming tubulin crystals, induces de nouo tubulin synthesis [30, 321. Interestingly, Moyer et al. showed that tubulin acts as a positive transcription factor for both vesicular stomatitis virus (VSV) and Sendai virus since a monoclonal antibody directed against the beta subunit of tubulin completely inhibited VSV and Sendai mRNA and virus RNA synthesis in extracts from infected cells or in detergent-disrupted virions [51]. In addition, these authors demonstrated that purified tubulin is required for the in vitro synthesis of Sendai virus mRNA and stimulates VSV mRNA synthesis in detergent-disrupted virions. Moreover, Hill et al. have shown that MT-associated proteins stimulate the in vitro transcription and replication of VSV [52]. These results suggest that tubulin and MT-associated proteins may be required transcriptional activating factors for these RNA viruses. It is tempting to speculate that free tubulin subunits may be involved in transcriptional regulation in the host cell which is directly related to cell progression through G,/G, or to the entry of the
DNA
43
SYNTHESIS
cell into the S phase of the cell cycle. If viruses, such as CMV, stimulate host cell DNA synthesis through a mechanism which involves alterations in the MT-tubulin equilibrium, then further elucidation of this mechanism may help in understanding events required to regulate both normal cell proliferation and oncogenic transformation of cells. REFERENCES 1.
Dulbecco, R. L., Hartwell, Acad. Sci. USA 53,403-410.
2.
Gershon, D., Sachs, L., and Acad. Sci. USA 56,918-925.
3
CELL
RESEARCH
19 1, 37-44
(1990)
Taxol Inhibits Stimulation of Cell DNA Synthesis by Human Cytomegalovirus REBECCA L. BALL,* DARRELL Departments
of ‘Microbiology
and PHuman
Biological
Chemistry
H. CARNEY,~ANDTHOMASALBRECHT**' and Genetics,
Inc.
INTRODUCTION Stimulation of cell DNA synthesis in quiescent cells appears to be a characteristic common to a number of transforming DNA viruses [l-5], and it has been suggested that stimulation of cell DNA synthesis is related to their oncogenic potential [6]. Human cytomegalovirus (CMV) infection of serum- or IUdR-arrested cells results in stimulation of cell DNA synthesis [5] and in an increase in the rate of cell division [7,8]. In addition, CMV infection has been associated with neoplastic disease [g-11], and it has been demonstrated that CMV malignantly transforms cultured hamster and human 1 To whom reprint Building, Route J-19, veston, TX 77550.
of Texas Medical
Branch,
Galveston,
Texas
77550
cells [12-141. Since stimulation of cell DNA synthesis may be related to the oncogenic potential of CMV and since a relationship between induction of a cellular proliferative signal and CMV replication has been proposed [15], it is important to determine the mechanism by which CMV initiates its mitogenic signal. A number of studies have suggested that altering the equilibrium between the microtubule (MT) polymer and unpolymerized tubulin can enhance or inhibit mitogenie signals. In some studies, colchicine, a MT-depolymerizing drug, blocks initiation of DNA synthesis [1621]. In other cases, increasing the level of unpolymerized tubulin subunits by colchicine treatment or microinjection ofphosphocellulose-purified tubulin subunits initiates DNA synthesis in serum-free fibroblast cultures [22; Carney, unpublished observations], or enhances stimulation of DNA synthesis in 3T3 cells treated with growth factors or hormones [23-261. Taxol, a drug which stabilizes intact MTs and stimulates polymerization of free tubulin, inhibits initiation of DNA synthesis in Go-arrested fibroblasts if added up to 8 h after thrombin or epidermal growth factor [27]. Together, these results suggest that a critical level of unpolymerized tubulin subunits may be required for cell progression through G,/G, and commitment to the S phase of the cell cycle. Alterations in the MT polymer could affect a number of early signal generating events in host cells including mobility of cell surface receptors or receptor interaction with signal generating molecules [see reference 28 for a review]. Other events which may occur later in the cell cycle, prior to entry into S phase, could also be affected. For example, a number of studies have shown that addition of growth factors to cells causes centriole duplication either at the Go/G, boundry or early in S [as]. In addition, the concentration of tubulin subunits has been shown to autoregulate the transcription and synthesis of tubulin [29-321. Thus, the extent of tubulin assembly plays a critical role in the regulation of new tubulin synthesis and, perhaps, in the synthesis of other molecules required for cell cycle progression. Experiments that have attempted to determine the temporal relationship between growth factor addition and MT-related events indicate that colchicine added
The microtubule (MT)-stabilizing drug, taxol, inhibited human cytomegalovirus (CMV)-initiated cell DNA synthesis by up to 100% in serum-arrested mouse embryo (ME) fibroblasts that were abortively infected by CMV. Taxol concentrations known to increase MT polymerization and to stabilize existing MTs (10 to 20 pg/ml) blocked CMV-stimulated cell DNA synthesis, while taxol concentrations of 2.5 pg/ml, or less, did not. Taxol maximally inhibited CMV initiation of cell DNA synthesis when added 3 h after virus infection and inhibited this initiation by greater than 50% when added up to 12 h after CMV infection. Control experiments suggest that taxol specifically inhibited CMV-stimulated cell DNA synthesis. Pretreatment of CMV stock with taxol did not reduce the stimulatory effect of CMV on cell DNA synthesis and taxol had no detectable effect on CMV-specific early protein synthesis. Moreover, taxol did not appear to alter thymidine pool sizes, affect cell viability, or compromise the DNA synthetic machinery in CMV-infected cells. Since taxol increases tubulin polymerization and inhibits MT disassembly, these results suggest that dynamic changes in MTs or in the pool of free tubulin subunits are necessary for CMV to stimulate cell entry into a proliferative cycle. (3 1990 Academic Press,
The University
requests should be addressed at Microbiology The University of Texas Medical Branch, Gal-
37
0014.4827/90
Copyright All rights
0
1990
of reproduction
by Academic in any form
$3.00
Press, Inc. reserved.
38
BALL,
CARNEY,
up to 10 h after growth factors can still enhance mitogenesis [23-261. In addition, in lymphocytes, where colchicine inhibits lectin-induced mitogenesis, this response is observed when colchicine is added up to 10 to 16 h after mitogen addition [ 16-171. These observations are consistent with our previous results showing that taxol can block initiation of DNA synthesis when added to cells up to 8 h after thrombin or epidermal growth factor [27]. In the present study, we have used taxol to examine if CMV initiates cell DNA synthesis by a mechanism that involves the equilibrium between free tubulin and the MT polymer. The present findings demonstrate that taxol blocks initiation of cell DNA synthesis by CMV in quiescent mouse embryo (ME) fibroblasts. These results suggest that the extent of MT polymerization or changes in the pool of unpolymerized tubulin subunits may be involved in regulating mitogenic signaling by CMV. Since previous studies have shown that other mitogenic signals involve dynamic changes in the MT-tubulin equilibrium, these studies suggest that such a MT-related signal may represent a common mechanism in growth factor and oncogenic virus stimulation of cell proliferation.
MATERIALS
AND
METHODS
Cell culture. Primary cultures of ME fibroblastic cells were obtained by trypsinization of the body wall of 9 to 13 day-old embryos as previously described [33]. ME cells were grown in 45% DulbeccoVogt’s modified Eagle’s medium, 45% Ham’s Fl2 medium (DV-F12 medium), 10% calf serum (growth medium), 100 U/ml penicillin, and 100 pg/ml streptomycin in a humidified atmosphere of 5% CO, in air at 37°C. Dulbecco-Vogt’s modified Eagle’s medium, Ham’s F12 medium, trypsin solution, glutamine, and antibiotics were obtained from Gibco (Grand Island, New York), and calf serum was purchased from Irvine Scientific (Irvine, CA). The derivation, culture, and maintenance of human embryonic thyroid (THY) and lung (LU) cells has been described in detail [34]. THY or LU cells were grown in 32 oz. glass prescription bottles in Eagle’s minimum essential medium (EMEM) supplemented with 10% y-irradiated fetal calf serum (yFCS) and 0.075% NaHCO,. Cell growth arrest. Quiescent cultures of ME cells were obtained by initially plating the cells at a density of 5 X lo* cells/cm* followed by incubation for 48 h in growth medium. The growth medium was then removed and replaced with medium without serum and incubation was continued for 10 min. The medium was changed to fresh serum-free medium and the cells were incubated at 37°C for 24 h. To provide a more synchronous population of cells, the cells were returned to growth medium for 2 h. Afterwards, the medium was removed, changed to serum-free medium as above, and the incubation in serum-free medium was continued for 48 h prior to virus infection. Virus propagation, infectivity assay, and control cell lysates. Strain AD169 of CMV was used for these studies and was propagated as described previously in detail [35]. When more than 90% of the cells demonstrated large nuclear inclusions for 3 days, the virus was harvested. Cells were scraped from the surface of the glass prescription bottles and the cells and debris were separated from the fluids by centrifugation at 184g. The clarified culture fluids were reserved. The pellet was resuspended in 5 ml of the reserved fluids and sonicated to lyse the cells. Debris was removed from the sonicated suspension by
AND
ALBRECHT
centrifugation as before and the clarified fluids were mixed with the reserved culture fluids (virus stock). Virus stocks were dispensed in 5 ml vials and stored at -70°C. Infectivities of the virus stocks were determined by plaque assay as previously described [34] and were in the range of 1 X lo6 to 2 X lo7 plaque forming units (PFU)/ml. Control mock-infected cell lysates were prepared from uninfected THY or LU cells in the same manner as virus stocks. CMV and mock infection. Serum-arrested ME cells in 35.mm dishes were infected with CMV as follows. The culture fluids were aspirated and the cells were inoculated with 0.5 ml of virus stock diluted in serum-free EMEM containing 0.075% NaHCO, to obtain a predetermined calculated multiplicity of infection. Control cultures were mock-infected with the same volume and dilution of cell lysate. The cells were incubated for 1 h at 37°C in 5% CO, with gentle rocking every 15 min to assure that the cells were constantly in contact with the inoculum. The inoculum was then aspirated and the cells were washed with serum-free DV-F12 and further incubated in fresh serum-free medium as described above (0 h postinfection, PI). Ultraviolet WV) irradiation. Virus stocks were UV-irradiated in 1 to 1.5 ml volumes with continuous stirring of the fluids. A GE-G8T5 bulb was used at a dose rate of 80 ergs/s/mm* as determined with a Blak-ray ultraviolet meter (Ultraviolet Products, Inc., San Gabriel, CA). Measurement of cell DNA synthesis. The effect of taxol on the mitogenic signal induced by CMV infection or mock infection was determined by measuring the rate of incorporation of [methyl-3H]thymidine ([3H]TdR, 20 Ci/mmol, ICN Radiochemicals, Irvine, CA) or [methyl-3H]hypoxanthine (10 Ci/mmol, New England Nuclear, Boston, MA) into cell DNA. [3H]TdR (1 &i/ml) was added at selected times after virus infection. At the end of each [3H]TdR pulse period, the cells were enzymatically digested and the DNA was released by the addition of 0.1% Protease XIV in 0.1X SSC (0.015 M NaCl, 0.0015 M Na citrate; pH 7.0), 2% sodium lauryl sarcosinate in 0.1X SSC, and 2% ethylenediaminetetraacetic acid (EDTA), and incubation at 37°C for 18 to 24 h [36]. Each sample was then titurated vigorously to shear the DNA, mixed thoroughly, and a 50 ~1 aliquot was applied to a 2.3 cm Whatman No. 3 filter paper disc to measure total DNA synthesis. DNA was precipitated onto the filter and filter papers were washed three times for 15 min each with cold 5% trichloroacetic acid (TCA). The filter papers were dried under a heat lamp or washed twice with ethanol and once with acetone, and air-dried overnight. Radioactivity was determined using 10 ml of 0.4% Omnifluor (New England Nuclear) in toluene. For labeling with [3H]hypoxanthine, an isotope concentration of 5 wCi/ml was used. Samples were treated after DNA extraction with 0.3 M KOH at 37°C for 12 h to hydrolyze labeled RNA before measurement of [3H]hypoxanthine incorporation into DNA as above [7]. Separation of uirus and cell DNA. The method of Crouch and Rapp [36] was used to separate virus and cell DNA in isopycnic gradients of CsCl. The DNA was centrifuged to equilibrium in CsCl gradients. The gradients were collected from the bottom of each tube in 8-drop fractions onto Whatman No. 3 filter paper discs. The radioactivity in each fraction incorporated into acid-precipitable material was determined as above. Samples were removed at regular intervals and the density was determined with an Abbe refractometer (Model B, Carl Zeiss, New York, NY). Autoradiography of labeled nuclei. The percentage of nuclei in which DNA was synthesized was determined by adding [3H]TdR (1 &i/ml) to CMVor mock-infected cells 24 h postinfection (PI). After a 6 h pulse, the cells were washed twice with PBS, fixed with 3% formaldehyde for 15 min at 4”C, washed with PBS, and extracted with cold 5% TCA for 15 min. The fixed cells were washed 3 times with PBS and allowed to air-dry. A thin film of Ilford K5 autoradiographic emulsion was applied to each dish and exposed for 2 weeks at ~20°C. The emulsion was developed and the nuclei stained with 2 ml of Hoeschst stain (5 wg/ml in PBS) for 30 min at 23°C. Nuclei that
CMV-INDUCED
CELL
DNA
-
incorporate [“HJTdR are covered with autoradiographic grains and all nuclei are fluorescent. Each culture was examined with a Leitz microscope with phase conkast and epifluorescence illumination. A minimum of 1500 labeled and unlabeled nuclei were scored for each determination. CelE ~~~bil~ty assay. At selected times PI, ME cells were removed from 35-mm dishes with trypsin solution. The cells were pelleted at 184g for 20 min. The supernatant fluids were decanted and the cell pellet was gently resuspended in trypan blue (10% in PBS). The percentage of viable (dye-excluding) cells in each sample was determined with the aid a hemocytometer. Immunofluorescence assay for CMV antigens. ME cells, grown on glass coverslips in Xi-mm dishes (2 X 105 cells/dish) were serumarrested and infected as described above. At selected times PI, the inoculum was aspirated and the cells were washed three times in PBS and fixed for 10 min in acetone (-20°C). Following fixation, the cetls were washed in PBS and incubated for 30 min at 37°C with human convalescent serum reactive to CMV strain AD169. The cells were then washed in PBS and incubated for 30 min with a fluorescein isothiocyanate-conjugated goat anti-human IgG. The cells were washed again in PBS and t,he coverslips were mounted on glass slides in PBS:glycerol (1:l). The cells were examined with a Zeiss fluorescence microscope. Drugs. Taxol was supplied by Dr. Matthew Suffness, National Products Branch, National Cancer Institute. Tax01 was disolved in ethanol to obtain a stock solution of 1.0 mg/ml and diluted with serum-free DV-F12 medium to achieve a final concentration of 10 @g/ml, unless indicated otherwise.
RESULTS
Absence
of HCMV
DNA
Synthesis
in ME Cells
Boldogh et al. [37] have reported that ME cells sustain an abortive infection with CMV in which CMV DNA synthesis is undetectable. Since the absence of CMV DNA synthesis in ME cells would facilitate this study by eliminating the need to separate cell and CMV DNA, we first attempted to confirm the findings of Boldogh and his colleagues. [3H]TdR-labeled DNA from CMV-infected ME cells and 14C-labeled DNA from mock-infected cells were ~osedimented to their buoyant densities in cesium chloride gradients. Consistent with the results of Boldogh et al. [37], synthesis of CMV DNA was not detected through 144 h PI in serum-arrested ME cells (data not shown). Furthermore, as previously reported by these investigators i37J, infectious yields of progeny CMV were not obtained from ME cells (data not shown). Thus, since CMV infection of ME cells (like infection of bovine, guinea pig, and hamster cells [7,38, 391) does not result in detectable synthesis of CMV DNA, the t3HJTdR incorporation observed in experiments with ME cells reflects cell DNA synthesis. Effect of Tax01 on Initiation by CMV
of Cell DNA Synthesis
To determine if the MT-tubulin equilibrium is involved in mitogenic signaling by CMV, serum-arrested ME cells were infected with CMV (1 PFUlcell) or were mock-infected with uninfected-cell lysate, and were
39
SYNTHESIS /
/
I
I
24
30
1
I
/
36
42
48
. CMV 0 CMV + taxoi A Lysate
A Lymte + tax01
LL..--~&.---li_..~ 0 6
12
18 Time
Post-lnfectlon
(h)
FIG. 1. Effect of taxol on CMV-initiated cell DNA synthesis. Serum-arrested ME cells were infected with 1 PFU/cell of CMV (circles) or were mock-infected (triangles) with the same amount and dilution of a cell lysate. Taxol(l0 pg/ml) was added at 3 h PI and was present for the duration of the experiment (open symbols). 13H]TdR (1 pCi/ml) was added for consecutive 6 h pulses through 48 h PI. Incorporation of 13H]TdR into acid-precipitable material was measured at the end of each pulse as described under Materials and Methods. Each dat,um point is plotted at the midpoint of the pulse period and represents the means of triplicate determinations.
treated with taxol (10 gg/ml) for the duration of the experiment or were left untreated. ~3H]TdR incorporation was measured for consecutive 6 h pulses through 48 h PI. As shown in Fig. 1, maximum stimulation of cell DNA synthesis by CMV was observed 24 to 30 h PI and was 2.5-fold greater than in control cultures. This stimulation was due to the presence of CMV since cells infected with UV-irradiated CMV stock (2.88 X lo5 ergs/mm’), which eliminated CMV infectivity, demonstrated levels of [3HJTdR incorporation comparable to those observed in cells mock-infected with uv-irradiated or with nonirradiated cell lysate (1.1 + 0.032 X 104 CPM for cells infected with irradiated CMV compared to 1.4 f 0.22 X lo4 or 1.4 ?z 0.043 X lo* for cells treated with nonirradiated or irradiated cell lysates, respectively). Taxol inhibited CMV-initiated cell DNA synthesis during this pulse period by about 75% suggesting that a pool of free tubulin subunits might be necessary for CMV to initiate a mitogenic signal. Influence of Taxol Concentration DNA Synthesis
on CMV-Initiated
Cell
The concentration dependence for tax01 inhibition of CMV-initiated cell DNA synthesis was determined by treating CMV- or mock-infected cultures with 0.3 to 20 pg/ml of taxol for the duration of the experiment and measuring 13H]TdR incorporation during a 6 h pulse from 24 to 30 h PI. As shown in Fig. 2, taxol concentrations of 2.5 pg/ml, or less, inhibited CMV-initiated cell DNA synthesis by less than 20%. In contrast, 10 or 20 pug/ml of taxol caused 100% inhibition of CMV-initiated
40
BALL,
CARNEY,
60 40 20
Taxol(pg/ml)
FIG. 2. The concentration effect of taxol on CMV-initiated cell DNA synthesis. The procedure was as described in the legend to Fig. 1 except that taxol was added at the concentrations indicated and [3H]TdR incorporation was determined 24 to 30 h PI. The data are derived from a representative experiment and are plotted as percent inhibition of CMV-stimulated [3H]TdR incorporation. The variation in the triplicate determinations used to calculate each datum point did not exceed 10%.
[3H]TdR incorporation, and half-maximal inhibition of CMV-induced cell DNA synthesis was observed at a taxol concentration of 6 pg/ml. These concentrations correspond to previously reported taxol concentrations required to prevent MT depolymerization in ME cells by colchicine [40, 411. Thus, these results suggest that taxol inhibition of CMV-stimulated DNA synthesis may be a direct result of taxol-induced alterations in the MT-tubulin equilibrium. The Time Course of Taxol Inhibition Cell DNA Synthesis
of CMV-Initiated
Because previous studies suggested that altered MTs might affect early events in cell entry into S phase, we investigated the time course for taxol sensitivity in CMV-infected ME cells. Taxol (10 pg/ml) was added at selected times after CMV infection (1 PFU/cell) and [3H]TdR incorporation was measured 24 to 30 h PI. Taxol added up to 9 h PI inhibited 70 to 100% of CMVstimulated cell DNA synthesis (Table 1). When added at 12 h PI, taxol caused only 50% inhibition suggesting that the taxol-sensitive event(s) had taken place in most cells by this time. Since taxol had little effect when added by 18 to 24 h PI, it appears that taxol did not nonspecifically impair the DNA synthetic machinery. In addition, at each of the time points shown, taxol caused an actual inhibition of CMV-initiated [3H]TdR incorporation and did not simply delay the onset of DNA synthesis (Fig. 1 and data not shown). Absence of a Direct Tax01 Effect on CMV To investigate the possibility that taxol might inactivate CMV, CMV stock was pretreated with taxol (10 pg/ml) at 37°C for 1 h. The virus stock was then diluted
AND
ALBRECHT
SO that a final calculated multiplicity of infection of 1 PFU/cell and a final taxol concentration of 0.5 pg/ml were achieved. As shown in Table 2, there was no significant difference in stimulation of [3H]TdR incorporation in serum-arrested ME cells infected with untreated CMV stock or with CMV stock pretreated with taxol. In the same experiment, however, 100% inhibition of CMV-initiated [3H]TdR incorporation was observed when 10 pg/ml of taxol was added 3 h PI. Accordingly, taxol did not exert its inhibitory effect on CMV-induced cell DNA synthesis by acting directly on the virus. To determine if taxol nonspecifically inhibited CMV early protein synthesis in ME cells, CMV-infected ME cells were treated with 10 pg/ml of taxol or were left untreated, and were examined by indirect immunofluorescence using human convalescent serum highly reactive to CMV antigens. No difference was noted in the percent of cells demonstrating immunofluorescence, in the location, or in the intensity of the staining of taxoltreated or untreated CMV-infected cells (data not shown), suggesting that taxol does not inhibit CMV early protein synthesis. These results also exclude the possibility that taxol had a substantial effect on adsorp-
TABLE Time
Time of tax01 addition relative to infection M 0
Course
of Taxol [3H]Thymidine
Viral-initiated [3H]thymidine incorporation (CPM t SD x IO-“)”
3 6 9 12
10.8 8.8 8.4 11.2 12.4
f k f + f
1.2 1.3 1.2 1.2 0.95
18
12.3 ? 1.3
24
11.7
4 0.51
1
Inhibition of CMV-Initiated Incorporation Viral-initiated [“Hlthymidine incorporation in the presence of taxol* (CPM + SD x lo-“) 4.4 k 0.82 0 1.4 2 0.19 3.3 t 0.53 5.8 * 0.35 9.9 T!c0.85 9.6 k 1.2
Inhibition (%)’ 60 100 83 70 53
19 18
o Values (CPM: counts per minute) represent the means of triplicate determinations from one representative experiment and are the difference in radioactivity for CMVand mock-infected cells in the absence of taxol treatment. The standard error of the difference = \/(SD,)‘/3 + @D&‘/3, w h ere SD, = standard deviation of the mean radioactivity for CMV-infected cells and SD, = standard deviation of the mean radioactivity for mock-infected cells. (1 Taxol (10 pg/ml) was added at the times indicated relative to removal of the inoculum (0 h) and was present for the duration of the experiment. [3H]thymidine (1 &i/ml) incorporation was determined 24 to 30 h PI. Values are the difference in radioactivity for taxoltreated, CMVand mock-infected cells. The standard error of the difference was calculated as described in footnote a. ’ Percent inhibition = [(V - L) - (V, ~ LT)] /V - L X 100, where V = radioactivity for CMV-infected cells, L = radioactivity for mock-infected cells, VT = radioactivity for CMV-infected cells treated with taxol, and L, = radioactivity for mock-infected cells treated with taxol.
CMV-INDUCED TABLE
TABLE
2
Effect of Pretreatment of CMV with Taxol on Virus Initiation of Cell DNA Synthesis Treatment CMV (1 PFU/cell) CMV pretreatment with taxol’ CMV with taxol’ added 3 h PI Cell lysate Cell lysate + taxol’
(CPM
Radioactivity f SEM X 10m4)”
3
Effect of Taxol on CMV Initiation of Cell DNA Synthesis as Determined by Measuring [3H]Hypoxanthine Incorporation
Stimulation of cell DNA synthesis (%jb
2.6 k 0.23
52
2.3 tr 0.14d
45
1.4 * 0.4Bd 1.7 -t 0.11 1.6 f 0.051
41
CELL DNA SYNTHESIS
0
’ Incorporation of [3H]thymidine (1 &i/ml) was determined 24 to 30 h PI. Values (CPM: counts per minute) represent the means of triplicate determinations from one representative experiment i standard error of the mean (SEM). b Percent stimulation of cell DNA synthesis = (V - L)/L X 100, where V = radioactivity for CMV-infected cells and L = radioactivity for mock-infected cells. ’ At 10 pg/ml, added to virus stock at 37°C 1 h prior to infection of ME cells. d Comparison of the data for untreated CMV and CMV pretreated with taxol by the Knuskal-Wallis and multiple comparison tests indicated that these numbers were not significantly different with an overall significance level of (Y = 0.05. Statistical analysis of the dat,a for cells infected with either untreated CMV or CMV pretreated with taxol indicated that these numbers were significantly different from the data for CMV-infected cells treated with taxol at 3 h PI or cells mock-infected with cell lysate. ‘At 10 pg/ml, added 3 h PI and present for the duration of the experiment.
tion or penetration of CMV. Therefore, taxol does not appear to affect CMV virions or synthesis of CMV early proteins. Evidence that Tax01 Effects on CMV-Stimulated DNA Synthesis Are Specific for Mitogenic Signal Events Studies of the specificity of taxol in ME cells have shown that taxol does not affect transport of small molecules, protein or RNA synthesis, cell morphology, or cell viability [27]. To exclude the possibility that taxol might nonspecifically affect CMV-infected ME cells, the following experiments were undertaken. First, to ensure that taxol caused a true inhibition of CMV-initiated [3H]TdR incorporation rather than inducing a change in thymidine pool sizes in CMV-infected ME cells, [3H]hypoxanthine was used to measure DNA synthesis. Taxol(l0 pglml) inhibited CMV-initiated cell DNA synthesis by 72% as measured by [3H]hypoxanthine incorporation (Table 3). Since the level of this inhibition is similar to the level of inhibition observed when [3H]TdR was used to measure DNA synthesis, taxol inhibition of CMV-induced cell DNA synthesis does not appear to be a consequence of a change in TdR pool sizes.
Radioactivity (CPM + SEM x 10m4)”
Treatment CMV CMV + taxold Cell lysate Cell lysate + taxold
12.3 9.4 7.7 8.1
k f zk f
0.12 1.0 0.38 0.24
Stimulation of cell DNA synthesis (%Y 62 16
Inhibition by taxol (%Y
72
’ Incorporation of [3H]hypoxanthine (5 pCi/ml) was determined 24 to 30 h PI. Values (CPM: counts per minute) represent the means of triplicate determinations from one representative experiment * standard error of the mean (SEM). * Percent stimulation of cell DNA synthesis = (V - L)/L X 100, where V = radioactivity for CMV-infected cells and L = radioactivity for mock-infected cells. ’ Percent inhibition = [(V - L) - (V, - L,)]/V - L X 100 where V = radioactivity for CMV-infected cells, L = radioactivity for mock-infected cells, V, = radioactivity for CMV-infected cells treated with taxol, and L, = radioactivity for mock-infected cells treated with taxol. dAt 10 pg/ml, added 3 h PI and present for the duration of the experiment.
Second, trypan blue exclusion was used to determine if cell death might account for the inhibition of CMV-initiated cell DNA synthesis by taxol. Cells were examined at 24 and 30 h PI for the ability to exclude dye (Table 4). As shown, taxol did not affect the viability of CMV-infected ME cells. Third, light microscopic examination showed no change in morphology of CMV-infected ME cells associated with taxol treatment. Fourth, we determined if taxol might inhibit the rate or extent of DNA synthesis in CMV-infected ME cells rather than the entry of cells into S phase. This distinction can be made by determining the actual number of cells synthesizing DNA. As shown in Table 5, the percentage of labeled
TABLE
4
Effect of Taxol on Cell Viability in CMV-Infected ME Cells Determined by Trypan Blue Exclusion Treatment CMV CMV + taxol* Cell lysate Cell lysate + taxol*
Viable cells (%)” 96.4 96.8 94.2 95.4
a Percent viable cells = dye-excluding cells/dye-excluding + stained cells X 100. A minimum of 250 cells were counted datum point. bAt 10 pg/ml, added 3 h PI and present for the duration experiment.
cells for each of the
42
BALL,
TABLE Effect of CMV-Infected Autoradiography
Taxol
on Mouse
Incorporation Embryo
CARNEY,
ANL)
5 of Cells
[“HjThymidine as Determined
in by
Labeled nuclei (%Y
Treatment CMV (1 PFU/cell) CMV + taxol’ Cell lysate Cell lysate + taxol’
21 9 8 6
o The percentage of labeled radiographically labeled and minimum of 1500 cells from b At 10 pg/ml, added 3 h experiment.
-
nuclei was determined by counting autononlabeled Hoechst-stained nuclei in a random fields. PI and present for the duration of the
nuclei determined by autoradiography was 2.5-fold greater in CMV-infected cultures than in control cultures. This increase correlates with the level of CMVstimulated [3H]TdR incorporation in ME cells. Thus, the CMV-induced stimulation of cell DNA synthesis represents an increase in the number of cells entering a proliferative cycle. Taxol (10 yglml) inhibited CMV-induced stimulation of nuclei labeling by nearly lOO%, confirming that taxol inhibits the initiation of cell DNA synthesis by CMV rather than the rate or extent of DNA synthesis.
DISCUSSION
The data presented in this report suggest that CMV, an oncogenic DNA virus, initiates cell DNA synthesis by a mechanism involving an alteration in the MT-tubulin equilibrium. Taxol inhibited CMV-initiated [3H] TdR incorporation by up to 100% suggesting that a critical concentration of free tubulin subunits, or changes in the extent of polymerization of existing MTs are required for mitogenic signaling by CMV. The concentration dependence of the taxol inhibition of CMV-initiated DNA synthesis parallels the concentration dependence for taxol stabilization of MTs and taxol inhibition of DNA synthesis stimulated by growth factors. Taxol concentrations of 2.5 pg/ml, or less, did not substantially inhibit initiation of t3H]TdR incorporation by CMV, while 10 or 20 pug/ml of taxol blocked CMV-initiated t3H]TdR incorporation. Previous studies in which MTs were visualized by antitubulin immunofluorescence [40] or experiments in which MT content was quantified by binding of a radioactive monoclonal antibody to tubulin [41] have shown that colchicine (1 PLM) depolymerizes most MTs in cells treated with
ALBRECHT
less than 5 Kg/ml of taxol. In contrast, 5 and 10 to 20 pug/ml of taxol partially and totally stabilize MTs against disruption by 1 pM colchicine, respectively. The taxol concentration dependence for inhibition of CMVinitiated cell DNA synthesis is similar also to the taxol concentration dependence for inhibition of initiation of DNA synthesis by growth factors. For example, taxol concentrations of less than 5 Fg/ml do not substantially inhibit thrombin-initiated DNA synthesis, while 5 wg/ ml and 10 to 20 pglml cause half-maximal and maximal inhibition, respectively [27]. Thus, the similarities in the concentration dependence for taxol inhibition of CMV- and growth factor-initiated DNA synthesis and inhibition of MT disruption by 1 yM colchicine suggest that these mitogenic agents share a common mechanism for initiation of cell DNA synthesis involving the MT-tubulin equilibrium. The time course for taxol inhibition of DNA synthesis also suggests that taxol inhibits an event(s) common to both growth factor and CMV initiation of cell proliferation. Taxol inhibited CMV initiation of cell DNA synthesis by 70 to 100% when added to quiescent ME cells up to 9 h after CMV infection. By 12 h PI taxol inhibited approximately 50% of the CMV-induced stimulation of cell DNA synthesis, and after this time had little effect. Previous studies have shown that taxol inhibits thrombin- or epidermal growth factor-initiated DNA synthesis in ME fibroblasts maximally when added during the first 6 hrs after the growth factor and is 50% as effective by 8 h [27]. Thus, taxol inhibits initiation events for a somewhat longer period of time after CMV infection than after growth factor treatment. The maximum rate of TdR incorporation after growth factor treatment of ME cells is from 20 to 24 h compared to 24 to 30 h for CMV stimulated cells. Accordingly, the time course of taxol sensitivity of CMV-stimulated cell DNA synthesis may be extended somewhat due to the requirement for synthesis of early CMV or cellular proteins which may be involved in mitogenic signaling [13,15]. The observation that taxol interrupts the proliferative signal generated by CMV and growth factors with similar timing suggests that both of these mitogenic agents may require changes in the MT-tubulin equilibrium during a critical period to commit cells to DNA synthesis. Although taxol appears to specifically block CMV initiation of t3H]TdR incorporation in a manner similar to its inhibition of growth factor-induced mitogenesis, it is not clear if this inhibition is due to a decrease in the pool of free tubulin and/or stabilization of preexisting MTs. Alternatively, taxol may restrict specific MT-directed events which might require the regulated polymerization of new tubulin subunits such as reorganization of the golgi apparatus and the splitting and maturation of centrioles. Interestingly, both of these events occur late
CMV-INDUCED
CELL
in G, prior to cell entry into S phase and correlate with the ability of cells to enter S phase [28, 421. A number of studies have demonstrated that MTs are disrupted or disorganized in transformed cells [43-481, leading to the hypothesis that a sustained increase in the level of unpolymerized tubulin may be related to uncontrolled proliferation in transformed cells. Pfeiffer et al. have shown that CMV infection results in MT depolymerization in some fibroblastic cells [49]. In our studies, although taxol-stabilized MTs appeared similar in CMV- and mock-infected cells, it was not possible to demonstrate a correlation between CMV stimulation of cell DNA synthesis and depolymerization of MTs (data not shown). To observe individual MTs by immunofluorescence with an acceptable level of background staining, low cell densities were required where neither growth factors nor CMV stimulate cell DNA synthesis effectively. Nevertheless, the lack of observable MT depolymerization in CMV-infected cells, when considered together with our findings for the taxol effect on CMVinitiated cell DNA synthesis, suggests that CMV-induced mitogenesis may require a critical free tubulin pool size or changes in functional subsets of MTs [50], rather than affecting gross changes in the total cytoplasmic MT complex. Recent evidence suggests that unpolymerized tubulin dimers may be directly involved in modulation of tubulin mRNA levels in cells. Ben-Ze’ev and Cleveland showed that tubulin autoregulates its synthesis indicating that the free tubulin pool size controls tubulin synthesis [29,30]. For example, tubulin synthesis is rapidly and specifically depressed in cells treated with drugs such as colchicine that increase free tubulin content [30] or in cells microinjected with exogenous tubulin [31]. In cont,rast, vinblastine, which decreases the free tubulin pool size by forming tubulin crystals, induces de nouo tubulin synthesis [30, 321. Interestingly, Moyer et al. showed that tubulin acts as a positive transcription factor for both vesicular stomatitis virus (VSV) and Sendai virus since a monoclonal antibody directed against the beta subunit of tubulin completely inhibited VSV and Sendai mRNA and virus RNA synthesis in extracts from infected cells or in detergent-disrupted virions [51]. In addition, these authors demonstrated that purified tubulin is required for the in vitro synthesis of Sendai virus mRNA and stimulates VSV mRNA synthesis in detergent-disrupted virions. Moreover, Hill et al. have shown that MT-associated proteins stimulate the in vitro transcription and replication of VSV [52]. These results suggest that tubulin and MT-associated proteins may be required transcriptional activating factors for these RNA viruses. It is tempting to speculate that free tubulin subunits may be involved in transcriptional regulation in the host cell which is directly related to cell progression through G,/G, or to the entry of the
DNA
43
SYNTHESIS
cell into the S phase of the cell cycle. If viruses, such as CMV, stimulate host cell DNA synthesis through a mechanism which involves alterations in the MT-tubulin equilibrium, then further elucidation of this mechanism may help in understanding events required to regulate both normal cell proliferation and oncogenic transformation of cells. REFERENCES 1.
Dulbecco, R. L., Hartwell, Acad. Sci. USA 53,403-410.
2.
Gershon, D., Sachs, L., and Acad. Sci. USA 56,918-925.
3
