Journal
of General Virology (1999), 80, 83–90. Printed in Great Britain
...................................................................................................................................................................................................................................................................................
Induction of human herpesvirus-8 DNA replication and
transcription by butyrate and TPA in BCBL-1 cells
Yimin Yu,1 Jodi B. Black,2 Cynthia S. Goldsmith,2 Philip J. Browning,3 Kapil Bhalla1, 4
and Margaret K. Offermann1, 4
1
Winship Cancer Center, Emory University, 1365-B Clifton Road NE, Atlanta, GA 30322, USA
Centers for Disease Control and Prevention, Atlanta, GA, USA
3
Vanderbilt Cancer Center, Vanderbilt University, Nashville, TN, USA
4
Division of Hematology/Oncology, Department of Medicine, Emory University, Atlanta, GA 30322, USA
2
Human herpesvirus-8 (HHV-8) is a gammaherpesvirus that is present primarily in a state of low
level persistence in primary effusion lymphoma cell lines. Using BCBL-1 cells that harbour HHV-8
but lack Epstein–Barr virus, we demonstrate that sodium butyrate is much more effective than the
phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) at inducing high levels of class II and
III virus transcription and viral DNA replication, but also initiates apoptosis. Apoptosis occurs prior
to assembly of virions when high concentrations of butyrate (1–3 mM) are used, whereas reduction
of butyrate concentration to 0n3 mM decreases the rate of apoptosis and results in production and
secretion of enveloped virions that are visualized at high number by electron microscopy in
approximately 20 % of BCBL-1 cells. Butyrate induces much higher levels of multiple class II and
class III transcripts than does TPA, including v-MIP I, v-IL-6, v-Bcl-2, vGPCR and ORF26. A decrease
in concentration of butyrate from 3 to 0n3 mM delays the peak induction of these genes, but peak
levels remain higher than peak levels in response to TPA. These studies indicate that the massive
apoptosis induced by 3 mM butyrate could be diminished and delayed by reduction of butyrate
concentration to 0n3 mM, thereby allowing expression of high levels of lytic-associated genes and
production of high yields of HHV-8 virions.
Introduction
Human herpesvirus-8 (HHV-8), also referred to as Kaposi’s
sarcoma-associated herpesvirus, is a gammaherpesvirus that
was originally identified in Kaposi’s sarcoma (KS) lesions from
human immunodeficiency virus (HIV)-infected individuals and
has since been found in KS lesions from all known risk groups
(Chang et al., 1994, 1996 ; Chang & Moore, 1996 ; Offermann,
1996 ; Rady et al., 1995). HHV-8 is also present primarily in a
latent state or in a state of low level persistence in primary
effusion lymphoma cells (Arvanitakis et al., 1996 ; Cesarman et
al., 1995 b ; Gaidano et al., 1996). These cells are derived from
a rare HIV-associated lymphoma that is characterized by the
development of lymphomatous effusions with little lymph
node involvement (Cesarman et al., 1995 a). Although HHV-8
is difficult to culture in cells derived from KS lesions, HHV-8
Author for correspondence : Margaret Offermann (at the Winship
Cancer Center). Fax j1 404 778 5016. e-mail mofferm!emory.edu
0001-5768 # 1999 SGM
can be maintained and induced to lytic replication in some cell
lines derived from primary effusion lymphomas (Arvanitakis et
al., 1996 ; Gaidano et al., 1996 ; Miller et al., 1996, 1997 ; Renne
et al., 1996 b). Although many primary effusion lymphoma cells
are co-infected with Epstein–Barr virus (EBV), some lines, such
as BCBL-1 cells, are infected only with HHV-8 (Renne et al.,
1996 b). The presence of HHV-8 in all reported cases of
primary effusion lymphoma suggests that HHV-8 may be
essential for the pathogenesis of this rare lymphoma.
In cells derived from primary effusion lymphomas, HHV-8
DNA appears to be predominantly in the nucleus in episomal
structures (Renne et al., 1996 a). The HHV-8 genome consists
of a 140n5 kb unique coding region flanked by multiple GCrich 801 bp terminal repeat sequences (Russo et al., 1996). At
least 81 open reading frames (ORFs) have been identified in the
HHV-8 genome, and most of the viral genes have sequence
and positional identity to known gammaherpesvirus genes
(Moore et al., 1996 a, b ; Russo et al., 1996). In addition, some
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Y. Yu and others
ORFs encode proteins that are homologous to cellular proteins,
including Bcl-2, Flice inhibitory protein (FLIP), interleukin-6
(IL-6), G-protein-coupled receptor (GPCR), cyclin D, interferon-regulatory factor and macrophage inflammatory proteins
(MIP) (Moore et al., 1996 a ; Russo et al., 1996). All of these
genes are located in non-conserved interblock regions of the
HHV-8 genome (Russo et al., 1996).
Although HHV-8 is primarily latent in most primary
effusion lymphoma cell lines, lytic replication can be induced
by either the phorbol ester 12-O-tetradecanoyl phorbol-13acetate (TPA) or by butyrate, agents that differ in mechanism
of action. Butyrate, but not TPA, preferentially induces
replication of HHV-8 over that of EBV, whereas TPA
preferentially induces EBV replication in BC-1 cells, a cell line
that is dually infected with HHV-8 and EBV (Miller et al.,
1997). Despite the greater responsiveness of HHV-8 to
butyrate than to TPA in BC-1 cells, a classification system for
HHV-8 gene transcription was recently developed based on
patterns of responsiveness to TPA in BC-1 cells (Sarid et al.,
1998). By this system, class I transcripts are those constitutively
expressed and not induced to higher levels by TPA and
presumably represent latent transcription ; class II transcripts
are expressed constitutively but are also induced to higher
levels by TPA ; class III transcripts are those that are not
constitutively expressed but are induced to high levels by
TPA, presumably representing genes responsible for lytic
replication.
In the current studies, we examine changes in DNA
replication, viral gene expression and virus production in
response to butyrate and TPA in BCBL-1 cells, a cell line
infected with HHV-8 but not with EBV. This cell line is known
to undergo lytic replication and production of virions in
response to TPA (Renne et al., 1996 b). We demonstrate that
butyrate is much more effective than TPA at inducing HHV-8
DNA replication and expression of class II and class III genes.
Butyrate, but not TPA, also induces apoptosis in a dosedependent manner. Although apoptosis occurs before virus
assembly in cells treated with high concentrations of butyrate,
the rate of apoptosis decreases when lower concentrations of
butyrate are used, and the enhanced cell survival facilitates
secretion of large numbers of enveloped HHV-8 virus.
Methods
Cell culture. BCBL-1 cells (NIH AIDS Research and Reference
Program, Rockville, MD, USA) were cultured in RPMI 1640 supplemented with 10 % foetal calf serum, 2 mM -glutamine, 100 U penicillin
and 100 U streptomycin. Cell density was maintained between 2i10&
and 10i10& cells\ml. At time 0, cells were collected by centrifugation at
1500 r.p.m. for 5 min and suspended in fresh medium at a density of
either 2i10&\ml or 5i10&\ml ; equal aliquots were then incubated with
either standard medium or medium supplemented with TPA (Sigma) or
sodium butyrate.
RNA isolation and Northern blotting. Total cellular RNA was
prepared using RNAzol B (Tel-Test) according to the manufacturer’s
recommendation. For Northern blot analysis, RNA (20 µg) was sizeIE
fractionated on a 1 % agarose–formaldehyde gel in the presence of
1 µg\ml ethidium bromide (Selden, 1987). The RNA was transferred to
nitrocellulose and covalently linked by both baking in vacuo for 2 h at
80 mC and ultraviolet irradiation using an ultraviolet cross-linker
(Stratalinker, Stratagene). Hybridizations were performed at 42 mC
overnight in 5iSSC (1i : 150 mM NaCl, 15 mM sodium citrate), 1 %
SDS, 5i Denhardt’s solution, 50 % formamide, 10 % dextran sulfate and
100 µg\ml sheared denatured salmon sperm DNA. Approximately
1–2i10' c.p.m.\ml labelled probe (spec. act. 10) c.p.m.\µg DNA) was
used in each hybridization. Following hybridization, membranes were
washed with a final stringency of 0n2i SSC in 0n1 % SDS at 55 mC. The
nitrocellulose was stripped using boiling water prior to rehybridization
with other probes. To avoid carryover contamination, membranes were
checked with a Geiger counter to ensure complete stripping and then
rehybridized with probe detecting transcripts with different size.
Autoradiography was performed with an intensifying screen at k70 mC.
Quantification was done with a scanning densitometer.
32P-labelling of probes. DNA probes were labelled with $#P
using oligolabelling kits (Pharmacia Biotech) according to the manufacturer’s recommendations. For v-cyclin, v-GPCR and v-MIP-I, the probes
consisted of the entire coding regions (Russo et al., 1996). For v-Bcl-2,
PCR using primers described by Moore et al. (1996 a) were used to
amplify fragments that were then radiolabelled with the oligolabelling
kit. The HHV-8 ORF26 transcript was detected by using a labelled
930 bp amplimer of ORF26 as described (Offermann et al., 1996).
DNA preparation and Southern blotting. Approximately
5i10' cells for each condition were lysed in 3 ml 2i lysis buffer
(40 mM Tris–HCl pH 7, 20 mM EDTA, 200 mM NaCl, 1 % SDS)
supplemented with 100 µg\ml proteinase K and then incubated at 65 mC
for 30 min followed by an overnight incubation at 37 mC. Cell lysates
were extracted twice with phenol. DNA was precipitated in 70 % ethanol.
DNA pellets were dissolved in 400 ml TE, and RNase A was added to a
final concentration of 100 µg\ml. Following incubation at 37 mC for 1 h,
RNase A was removed by phenol extraction. After precipitation with
70 % ethanol, DNA pellets were dissolved in TE. DNA samples were
digested with restriction enzymes, and DNA was separated by 1 %
agarose gel and transferred to nitrocellulose membrane. Cross-linking
and hybridizations were as described for Northern blot analysis.
Extracellular HHV-8 virion preparation. BCBL-1 cells were
pelleted by centrifugation at 400 g for 5 min. The cell-free culture
medium was collected and centrifuged at 3500 g for 30 min to remove
cellular debris, and virus was then pelleted from the medium by
centrifugation at 15 000 g for 4 h. Pellets were resuspended in resuspension buffer (40 mM Tris–HCl pH 7, 10 mM NaCl, 6 mM MgCl , 10 mM
#
CaCl ). DNase and RNase (20 U) were added followed by incubation at
#
37 mC for 1 h to eliminate free DNA and RNA. DNase was inactivated by
incubating at 65 mC for 10 min. An equal volume of lysis buffer
supplemented with proteinase K (100 µg\ml) was added, and the
solution was incubated at 65 mC for 30 min followed by 37 mC overnight.
After phenol–chloroform extraction and ethanol precipitation, DNA
pellets were dissolved in TE and subjected to restriction digestion and
Southern blotting.
For detergent sensitivity, virus pellets were resuspended in TE with or
without NP-40 at a final concentration of 1 %, and the mixture was
incubated at 37 mC for 30 min. Proteinase K was added to a final
concentration of 0n5 mg\ml and incubated at 37 mC for 30 min. MgCl
#
was added to 5 mM with 20 U DNase I and incubated for a further 1 h
at 37 mC followed by the addition of 1\5 vol. 5i lysis buffer with
proteinase K (final concentration 1 mg\ml). Reactions were incubated at
65 mC for 30 min followed by 37 mC overnight. DNA was then isolated
and analysed as described above.
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HHV-8 induction by TPA and butyrate
Determination of cell number and cell viability. Changes in
cell number were determined by counting with a haemocytometer at the
indicated days. No additions or changes were made to the medium after
day 0. Percentage viability was determined by assessing the percentage
of cells that were able to exclude 0n1 % trypan blue.
Electron microscopy. Thin section electron microscopy was
performed as previously described (Goldsmith et al., 1995).
Flow cytometric analysis of apoptotic cells. After incubation
with or without butyrate or TPA, 1i10' cells from each condition were
fixed and assayed for DNA fragmentation using the APO-Direct kit
(Pharmingen) according to the manufacturer’s recommendation. This
assay is based on the labelling of DNA fragments found in apoptotic cells
using terminal deoxytransferase enzyme with fluorescein isothiocyanateconjugated dUTP (FITC-dUTP). Flow cytometric analysis was done
analysing 10 000 cells using a FACScan (Becton Dickinson).
Results
Induction of HHV-8 DNA replication by TPA and
butyrate
A range of concentrations of TPA and butyrate were
examined for their ability to induce HHV-8 DNA replication in
BCBL-1 cells. When examined at 2 days, TPA at concentrations
ranging from 20 to 200 ng\ml led to twofold induction of
intracellular HHV-8 DNA compared to control (Fig. 1 A). The
increase in response to 0n3 mM butyrate was comparable to
the increase that resulted from TPA, whereas 3 mM butyrate
led to a greater than sixfold increase. When examined at 5
days, the increases in intracellular viral DNA in response to
TPA (20–200 ng\ml) and butyrate (0n3 mM) were at least
seven- and ninefold greater, respectively, in comparison to
control cells (Fig. 1 B). Cells incubated with 3 mM butyrate
suffered massive cell death, precluding analysis of intracellular
HHV-8 DNA under this condition.
We next compared the ability of butyrate and TPA to
induce virus production and secretion. Cell-free culture medium
from control BCBL-1 cells and from cells incubated with TPA
or butyrate for 5 days were assessed for the presence of viral
DNA. Comparable amounts of DNase-resistant HHV-8 DNA
were present in medium from BCBL-1 cells incubated with TPA
at either 20 or 200 ng\ml, whereas incubation with 0n3 mM
butyrate led to higher amounts of DNase-resistant HHV-8
DNA than resulted from incubation with TPA at any
concentration (Fig. 1 C). In contrast, massive cell death occurred
in response to incubation with 3 mM butyrate, and conditioned
medium from these cells contained only trace DNase-resistant
HHV-8 DNA. The HHV-8 DNA that was induced by 0n3 mM
butyrate was protected from DNase digestion by both protein
and lipid. The DNA remained resistant to DNase in the
presence of proteinase K unless it was also treated with NP-40
(Fig. 1 D) ; this is indicative of enveloped virus. The production
of HHV-8 virions by BCBL-1 cells in response to incubation
with 0n3 mM butyrate for 5 days was confirmed by electron
microscopy. Viral nucleocapsids accumulated within a dense
granular material in cell nuclei (Fig. 2 A). Also present in some
cells were nuclear inclusions of viral core-like material,
marginated chromatin and a duplication of the nuclear
membrane. Nucleocapsids passed through the perinuclear
space into the cytoplasm and appeared to acquire their
envelope by budding upon the membranes of the Golgi
apparatus or the plasma membrane. Mature particles contained
a layer of tegument between the nucleocapsid and the envelope
(Fig. 2 B). Virus particles were detected in approximately 20 %
of cells (data not shown).
Toxicity in response to butyrate and TPA in BCBL-1
cells
Although butyrate enhanced HHV-8 DNA replication in a
dose-dependent manner, it was extremely toxic to cells when
used at high concentrations. Analysis of changes in cell number
and viability were undertaken to characterize these effects. Cell
number increased linearly for control cells between days 1 and
Fig. 1. Induction of HHV-8 DNA replication by TPA and butyrate. At time 0, BCBL cells were resuspended in fresh medium at a
density of 2i105 or 5i105 cells/ml and were incubated with either butyrate or TPA for the indicated times. DNA was purified
either from whole cells (A, B) or from conditioned medium (C, D). DNA was digested with the indicated restriction enzyme,
size fractionated on a 1 % agarose gel, and analysed by Southern blot analysis using a probe specific for HHV-8 ORF26. (A, B)
DNA (2 µg) prepared from cells treated with different concentrations of TPA or sodium butyrate for 2 days (A) or 5 days (B)
was digested with SacI and analysed by Southern blot analysis using radiolabelled ORF26. (C) Cell-free culture medium
(3n5 ml) was collected after 5 days of incubation and centrifuged to pellet virus particles as described in Methods. Pellets were
resuspended and incubated with DNase in order to eliminate any non-protected free viral DNA. DNase was heat-inactivated,
and DNA was purified, digested with HindIII and analysed by Southern blot analysis using ORF26 probe. (D) The pellet
obtained from cell-free medium of cells incubated with 0n3 mM butyrate for 5 days was incubated with DNase and proteinase K
with or without prior treatment with NP-40 as described in Methods. DNA was then isolated and analysed by Southern blotting.
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Y. Yu and others
Fig. 2. Electron microscopy of HHV-8 virions in BCBL-1 cells incubated with 0n3 mM butyrate for 5 days. (A) HHV-8
nucleocapsids (filled arrows), viral core-like material (open arrows) and duplicated nuclear membranes (curved arrows) are
present in the nucleus of this infected cell. Extracellular particles surround the cell (arrowheads). Bar, 1 µm. (B) Extracellular
HHV-8 particles consist of the nucleocapsid (filled arrow), surrounded by the tegument (curved arrow) and viral envelope
(arrowhead). Note that the tegument apparently adheres to the viral envelope in some particles, and that the envelope is
sometimes ‘ tailed ’. Bar, 100 nm.
4, whereas butyrate at either 3 mM or 1n5 mM completely
prevented the increase in cell number that occurred in control
cells (data not shown). Both TPA (20 ng\ml) and butyrate at
0n3 mM decreased the rate of increase in cell number of BCBLIG
1 cells, so that after 4 days of culture, cell number was 60 %
lower in cells incubated with either agent compared to control
BCBL-1 cells. Despite the comparable increase in cell number,
incubation with butyrate, but not with TPA, compromised cell
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HHV-8 induction by TPA and butyrate
response to butyrate. After a 24 h incubation, more that 50 %
of cells had significant DNA fragmentation in response to
3 mM butyrate, whereas there was no significant DNA
fragmentation detected by this assay at 24 h in response to
0n3 mM butyrate or to TPA (Fig. 3 B). When the percentage of
apoptotic cells was examined as a function of time, it was found
that a decrease in butyrate concentration from 3 mM to
0n3 mM dramatically delayed the onset and magnitude of
DNA fragmentation (Fig. 3 B). There was not significant DNA
fragmentation in response to 0n3 mM butyrate until day 4, at
which time 36 % of the cells incubated with 0n3 mM butyrate
showed DNA fragmentation. In contrast, only 7 % of BCBL-1
cells incubated with TPA for 4 days displayed measurable
DNA fragmentation. These data indicate that butyrate induced
apoptosis in BCBL-1 cells in a dose-dependent manner with
massive apoptosis apparent after 24 h of incubation with
3 mM butyrate, and reduction of butyrate concentration was
an effective way of decreasing the magnitude of the apoptosis
and enhancing cell survival.
Induction of HHV-8-encoded mRNAs by TPA and
butyrate
Fig. 3. Effect of TPA and butyrate on BCBL-1 cell viability and DNA
fragmentation. (A) BCBL-1 cells were grown in standard medium or
medium supplemented with TPA or butyrate at the indicated
concentrations. Percentage viability was determined as a function of time
by trypan blue exclusion. (B) BCBL-1 cells were incubated in complete
medium without supplement (control) or containing TPA or butyrate at the
indicated concentration. DNA fragments were labelled using terminal
deoxytransferase and FITC-dUTP with analysis by flow cytometry as
described in Methods. The M1 window was based on the fluorescence
peak that contained 99 % of control cells, whereas the M2 window
quantified cells that exceeded the amount of DNA fragments found in
control cells. The time-course analysing the percent apoptotic cells as
determined by the M2 window is plotted for TPA compared to butyrate.
viability. When examined at day 4, a comparable percentage of
the control and TPA-treated cells were viable (82 and 84 %,
respectively), whereas only 20 % of the cells treated with either
1n5 or 3 mM butyrate were viable (Fig. 3 A). When the
butyrate concentration was decreased to 0n3 mM, a higher
percentage of cells remained viable (64 %), but the percentage
was still considerably less than the percentage that occurred in
cells incubated with TPA. Higher concentrations of TPA
(200 ng\ml) did not decrease viability compared to cells
incubated with 20 ng\ml TPA (data not shown). Flow
cytometric analysis of DNA fragmentation was used to
investigate the role of apoptosis in cell death that occurred in
The kinetics and magnitude of induction of HHV-8encoded genes by TPA and butyrate were compared. When
concentrations of TPA and butyrate that were associated with
peak responses were used, peak levels of the class II transcripts
v-MIP I, v-IL-6 and the class III transcripts v-GPCR and v-Bcl2 were at least fourfold higher in response to butyrate than in
response to TPA (Fig. 4 A). Furthermore, peak levels occurred
by 24 h in response to butyrate but did not occur until 48 h in
response to TPA. The class I transcript, v-cyclin, was detectable
in unstimulated cells and also showed a minor transient
increase in response to both TPA and butyrate. The kinetics
and magnitude of the induction of v-cyclin mRNA differed
dramatically from the class II and III transcripts, with induction
peaking by 4 h in response to both TPA and butyrate and
declining more rapidly in response to butyrate than to TPA.
Induction of ORF26, a class III transcript expressed late in the
lytic phase, occurred much later than any of the other
transcripts examined. Elevated levels were detectable after
36 h incubation with either butyrate or TPA, and the levels
were more than tenfold higher in response to butyrate
compared to TPA. Only the most abundant transcript for each
probe is shown in Fig. 4 (A). Less abundant, higher molecular
mass transcripts consistent with known polycistronic patterns
of gene expression were also detected with probes for v-cyclin,
v-MIP I, v-GPCR, v-Bcl-2 and ORF26, several of which are
shown in Fig. 4 (C). In general, changes in the higher molecular
mass forms were similar to changes in the major transcripts
shown in Fig. 4 ; however, detailed analysis of these was not
undertaken to examine possible changes in mRNA processing.
The cellular gene glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) was used as a control and showed a decrease in
expression in response to butyrate but not to TPA.
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Y. Yu and others
Fig. 4. Induction of HHV-8 mRNA expression by TPA or sodium butyrate. BCBL-1 cells were incubated under control conditions
or with TPA or butyrate for the indicated times. Total cellular RNA (20 µg) was size-fractionated and analysed by Northern blot
analysis as described in Methods. The blot was serially probed for the indicated genes. The dominant band for each of the
genes indicated is shown in (A) with its estimated molecular mass. The time-course of induction using concentrations of TPA
(20 ng/ml) and butyrate (3 mM) associated with peak responses is shown in (A). Dose-response analysis at 24 h is shown in
(B). An analysis at late time-points is shown (C) in response to a low concentration of butyrate (0n3 mM) compared to the
concentration of TPA that leads to peak induction (20 ng/ml).
The kinetics of induction of class II and III transcription, but
not the magnitude of response, were dramatically altered when
the concentration of butyrate was reduced from 3 mM to 0n3
mM. Neither v-GPCR nor v-Bcl-2 was induced by 0n3 mM
butyrate at 24 h, whereas both of these mRNAs were induced
to high levels of expression by 3 mM butyrate at this same
time-point (Fig. 4 B). Although no induction by 0n3 mM
butyrate was seen at 24 h, high levels of v-GPCR and v-Bcl-2
were seen in response to incubation with 0n3 mM butyrate for
4 and 5 days (Fig. 4 C). In contrast to butyrate, higher levels of
TPA decreased the induction of v-GPCR (Fig. 4 B). Furthermore, examination up to 5 days showed only a marginal
induction of v-Bcl-2 by TPA at time-points when high levels of
v-Bcl-2 were induced by both 3 mM and 0n3 mM butyrate
(Fig. 4 A and C, respectively). The late lytic gene ORF26 was
also more effectively induced by butyrate than by TPA at all
concentrations examined (Fig. 4 A, C). The induction of ORF26
by butyrate occurred later in response to 0n3 mM compared to
3 mM, with high levels of induction in response to 0n3 mM
butyrate not occurring until day 4 (Fig. 4 C).
Discussion
In these studies, we demonstrate that butyrate at concentrations ranging from 0n3 to 3n0 mM induced much higher levels
of transcription of HHV-8 class II and class III genes and
greater amounts of DNA replication in BCBL-1 cells than did
TPA at concentrations ranging from 20 to 200 ng\ml. Butyrate
at 3 mM also induced massive apoptosis that occurred prior to
II
the production of virions. A tenfold reduction in butyrate
concentration to 0n3 mM delayed both apoptosis and the
expression of HHV-8 class II and class III transcripts. Despite
this delay, peak levels of class II and class III transcripts
remained considerably higher and were associated with greater
viral DNA production than occurred in response to TPA.
Furthermore, the decrease in the rate and magnitude of
apoptosis that occurred as a consequence of the reduction of
butyrate concentration was associated with the secretion of
mature, enveloped virus.
It is somewhat paradoxical that butyrate was the agent that
induced both the highest levels of v-Bcl-2 and the greatest
amount of apoptosis. Although v-Bcl-2 has been shown to
suppress Bax-mediated toxicity in both human and yeast cells
(Cheng et al., 1997 ; Sarid et al., 1997), the large increases in vBcl-2 mRNA in response to 3 mM butyrate occurred after the
apoptotic pathway was well under way. A decrease in butyrate
concentration to 0n3 mM dramatically reduced the rate of
apoptosis, but it also delayed the induction of v-Bcl-2. Thus,
much of the enhanced cell survival and virion production
associated with the lower concentration of butyrate occurred
prior to the induction of v-Bcl-2. Furthermore, cells incubated
with TPA did not have any evidence of apoptosis until day 4,
even though v-Bcl-2 mRNA levels were barely detectable. It is
thus unclear whether the induced v-Bcl-2 had any role in the
inhibition of apoptosis and production of virions in these cells.
Our results with BCBL-1 cells are consistent with those seen in
BC-1 cells, which also undergo more HHV-8 DNA replication
in response to butyrate than in response to TPA (Miller et al.,
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HHV-8 induction by TPA and butyrate
1997). The majority of BC-1 cells incubated with 3 mM
butyrate were reported to lyse prior to production of
encapsidated HHV-8 virus, but it remains to be determined
whether a dose response to butyrate occurs in BC-1 cells that
is similar to the one we report in BCBL-1 cells.
The transient induction of v-cyclin that occurred in
response to TPA and butyrate was unexpected. Previous
reports on v-cyclin have reported that it is insensitive to
induction with TPA when examined at 48 h ; therefore, it is
classified as a class I gene (Sarid et al., 1998). The induction of
v-cyclin that we observed occurred early, and v-cyclin mRNA
levels were at or lower than baseline by 12 h and 24 h in
response to butyrate and TPA, respectively. Furthermore, the
magnitude of the induction of this class I gene was low
compared to the magnitude of induction of the class II and class
III genes. Thus, our data are consistent with the classification of
v-cyclin as a class I transcript. The 2n0 kb transcript seen in Fig.
4 (A) with the v-cyclin probe is bicistronic and encodes both vcyclin (ORF72) and v-FLIP (ORF71) (Rainbow et al., 1997).
This transcript originates upstream of ORF73 (LANA) from
the same promoter used for ORF73 and splices out the
complete ORF73. Our Northern blots lacked the sensitivity to
determine whether butyrate or TPA altered splice patterns for
these transcripts, but no obvious changes in the transcripts
hybridizing with the v-cyclin probes were detected to suggest
alternative splicing. The v-cyclin was the only HHV-8 gene
examined that showed less response to butyrate than to TPA,
consistent with butyrate being a more effective inducer of lytic
replication. It was nonetheless surprising that its levels did not
decline when cells began to express class II and III transcripts.
These studies demonstrate that the toxicity associated with
the use of butyrate in BCBL-1 cells can be largely overcome by
a reduction in concentration to one that decreases the rate of
apoptosis while maintaining the ability of butyrate to induce
higher levels of class II and class III transcription than result
from using TPA. The lower concentrations of butyrate delay
the peak induction of class II and III transcripts but are
nonetheless associated with higher peak levels than occur in
response to TPA. These studies thus define conditions that
overcome many of the difficulties associated with induction of
lytic replication by butyrate and offer an alternative to TPA
that may helpful for production of HHV-8 virions and for
studies on changes from latent to lytic replication of HHV-8.
We would like to thank Dr Philip Pellet for helpful discussions and for
critical review of the manuscript. This work was supported in part by the
Winship Cancer Center of Emory University and by NIH grants RO1
CA67382 and P30AR42687.
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Received 19 June 1998 ; Accepted 2 September 1998
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