---
id: "edwards-2011-geranylgeranyl"
title: "Inhibition of Geranylgeranyl Transferase-I Decreases Cell Viability of HTLV-1-Transformed Cells"
authors:
  - "Dustin C. Edwards"
  - "Katherine M. McKinnon"
  - "Claudio Fenizia"
  - "Kyung-Jin Jung"
  - "John N. Brady"
  - "Cynthia A. Pise-Masison"
venue: "Viruses"
year: 2011
date: "2011-10-10"
doi: "10.3390/v3101815"
url: "/research/publications/10-3390-v3101815/"
pdf: "/research/publications/10-3390-v3101815/dustin-edwards-10-3390-v3101815.pdf"
pmc: "https://pmc.ncbi.nlm.nih.gov/articles/PMC3205383/"
openAccess: true
license: "cc-by"
citedBy: 5
citedBySource: "OpenAlex, read 2026-09-12"
---
# Inhibition of Geranylgeranyl Transferase-I Decreases Cell Viability of HTLV-1-Transformed Cells

A geranylgeranyl transferase inhibitor reduces viability of HTLV-1-transformed cells and lowers Tax.

## Abstract

Human T-cell leukemia virus type-1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL), an aggressive and highly chemoresistant malignancy. Rho family GTPases regulate multiple signaling pathways in tumorigenesis: cytoskeletal organization, transcription, cell cycle progression, and cell proliferation. Geranylgeranylation of Rho family GTPases is essential for cell membrane localization and activation of these proteins. It is currently unknown whether HTLV-1-transformed cells are preferentially sensitive to geranylgeranylation inhibitors, such as GGTI-298. In this report, we demonstrate that GGTI-298 decreased cell viability and induced G2/M phase accumulation of HTLV-1-transformed cells, independent of p53 reactivation. HTLV-1-LTR transcriptional activity was inhibited and Tax protein levels decreased following treatment with GGTI-298. Furthermore, GGTI-298 decreased activation of NF-κB, a downstream target of Rho family GTPases. These studies suggest that protein geranylgeranylation contributes to dysregulation of cell survival pathways in HTLV-1-transformed cells.

## Full text

Machine-extracted from the PDF linked above. It carries the artifacts that come with reading a typeset two-column page: running heads, figure captions in the flow of the prose, and words broken across line ends. The abstract above is the registry's deposit and is the authoritative text.

Viruses 2011, 3, 1815-1835; doi:10.3390/v3101815
viruses
ISSN 1999-4915
www.mdpi.com/journal/viruses
Article
Inhibition of Geranylgeranyl Transferase-I Decreases Cell
Viability of HTLV-1-Transformed Cells
Dustin C. Edwards 1, Katherine M. McKinnon 2, Claudio Fenizia 2, Kyung-Jin Jung 1,
John N. Brady 1,† and Cynthia A. Pise-Masison 1,*
1 Virus Tumor Biology Section, Laboratory of Cellular Oncology, Center for Cancer Research,
National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA;
E-Mails: edwardsd2@mail.nih.gov (D.C.E.); jungk@kitox.re.kr (K.-J.J.)
2 Vaccine Branch, Center for Cancer Research, National Cancer Institute, National Institutes of
Health, Bethesda, MD 20892, USA; E-Mails: mckinnonkm@mail.nih.gov (K.M.M.);
feniziac@mail.nih.gov (C.F.)
† Deceased.
* Author to whom correspondence should be addressed; E-Mail: masisonc@mail.nih.gov;
Tel.: +1-301-435-2499; Fax: +1-301-496-4951.
Received: 13 September 2011 / Accepted: 26 September 2011 / Published: 10 October 2011
Abstract: Human T-cell leukemia virus type-1 (HTLV-1) is the etiological agent of adult
T-cell leukemia (ATL), an aggressive and highly chemoresistant malignancy. Rho family
GTPases regulate multiple signaling pathways in tumorigenesis: cytoskeletal organization,
transcription, cell cycle progression, and cell proliferation. Geranylgeranylation of Rho
family GTPases is essential for cell membrane localization and activation of these proteins.
It is currently unknown whether HTLV-1-transformed cells are preferentially sensitive to
geranylgeranylation inhibitors, such as GGTI-298. In this report, we demonstrate that
GGTI-298 decreased cell viability and induced G2/M phase accumulation of HTLV-1-
transformed cells, independent of p53 reactivation. HTLV-1-LTR transcriptional activity
was inhibited and Tax protein levels decreased following treatment with GGTI-298.
Furthermore, GGTI-298 decreased activation of NF-κB, a downstream target of Rho
family GTPases. These studies suggest that protein geranylgeranylation contributes to
dysregulation of cell survival pathways in HTLV-1-transformed cells.
OPEN ACCESS

Viruses 2011, 3 1816
Keywords: human T-cell leukemia virus type-1; HTLV-1; Tax; long terminal repeat; LTR;
geranylgeranyltransferase; GGTI-298; small GTPase; NF-κB; p53; cell cycle
1. Introduction
An estimated 10–20 million people worldwide are infected with human T-cell leukemia virus
type-1 (HTLV-1) [1], the etiological agent of adult T-cell leukemia (ATL) [2,3] and tropical spastic
paraparesis/HTLV-1 associated myelopathy (TSP/HAM) [4–6]. ATL is characterized as a clonal
lymphoproliferative disorder of CD4+ T-lymphocytes. Approximately 3 to 5% of HTLV-1-infected
individuals develop ATL, which presents after a long period of clinical latency that can span several
decades [7]. Disease onset is consistent with a multi-step process of T-lymphocyte immortalization
and transformation. The mechanism by which infected individuals develop ATL is unknown, however
the 40 kDa viral oncoprotein, Tax, has been associated with cellular transformation [8].
HTLV-1 Tax is essential for viral gene expression and Tax disrupts pathways involved in cell cycle
regulation, apoptosis, and DNA damage responses through direct interaction with regulatory proteins
and regulation of nuclear factor κB (NF-κB), cyclic AMP response element and activating
transcription factor-1 (CRE/ATF-1), and serum response element (SRE) transcription pathways [8,9].
NF-κB transcriptional activation is triggered by phosphorylation of the upstream IκB kinase (IKK)
complex through the phosphoinositide 3-OH kinase (PI3K)/Akt pathway [10]. Interestingly,
phosphorylation of the NF-κB p65/RelA subunit at serine 536 contributes to the transcriptional
inhibition of p53 in Tax-expressing cells. In HTLV-1-transformed cells, inhibition of p53 by Tax has a
role in cell growth, cell cycle progression, and inhibition of apoptosis [11]. It has been shown
that several chemical compounds induce apoptosis by reactivating p53 in HTLV-1-transformed
cells [12–14]. Reactivation of p53, or induction of apoptosis in a p53-independent manner are
therefore ideal strategies for the development of drugs to preferentially target ATL.
A new class of anticancer drugs has been recently demonstrated to decrease cell viability in
multiple transformed cell lines through a p53-independent mechanism by inhibiting geranylgeranyl
transferase 1 (GGTase-1) [15–17]. Geranylgeranyl transferase inhibitors (GGTIs), such as GGTI-298,
inhibit a range of geranylgeranylated proteins, including the Rho family of small GTPases: Rho, Rac,
and Cdc42 [18]. Activation of NF-κB by the Rho family has been well documented and GGTI-298 has
been shown to affect the NF-κB pathway [17,19]. The effects of GGTI-298 are likely to be specific to
cell type, external stimuli, and species [19,20]. In addition to regulation of NF-κB, Rho family
members have been shown to regulate cellular gene transcription through CREB, SRF, and c-Jun
transcription factor binding sites, each of which are present within Tax-responsive elements of the
HTLV-1 promoter and have a role in viral gene expression [21–25]. Determination of whether
HTLV-1-transformed cells are sensitive to geranylgeranyl transferase inhibitors would advance our
understanding of how geranylgeranylated proteins regulate cell survival in these cells.
In this report we provide evidence that treatment of HTLV-1-transformed cells with GGTI-298
caused a significant decrease in cell viability. GGTI-298 induced G2/M phase accumulation and
inhibited NF-κB in these cells. In contrast to other small molecule inhibitors, GGTI-298-mediated

Viruses 2011, 3 1817
inhibition of NF-κB did not reactivate p53. GGTI-298 decreased Tax expression and transcriptional
activation of the HTLV-1-LTR. Moreover, the decreased phosphorylation of IκB in GGTI-298-treated
HTLV-1-transformed cells did not correlate with Tax protein levels. These studies suggest that protein
geranylgeranylation contributes to dysregulation of cell survival pathways in HTLV-1-transformed cells.
2. Results and Discussion
2.1. GGTI-298 Decreases the Viability of HTLV-1-Transformed Cells
We first sought to determine whether inhibitors of farnesyl transferase (FTase) or geranylgeranyl
transferase I (GGTase I), catalysts of small GTPase prenylation, affect the viability of HTLV-1-
transformed cells. Membrane association is critical for small GTPase activation and is mediated by
covalent addition of C15 farnesyl or C20 geranylgeranyl isoprenyl groups to cysteine residues in the
CAAX tetrapeptide motif near or at their carboxyl terminus [26]. Ras GTPases are modified by
farnesylation while the majority of Rho family GTPases are geranylgeranylated. To examine
sensitivity to prenylation inhibitors, HTLV-1-transformed cell lines C8166, C91/PL, and MT2 and
control PBMCs from three different donors were treated with DMSO solvent, 20μM FTI-277 or
10 μM GGTI-298 (concentrations routinely used for other cancer types). As PBMCs were cultured in
the presence of interleukin-2 (IL-2), HTLV-1-infected SP cells cultured in the presence of IL-2 served
as an additional control to determine whether IL-2 contributed to the survival of drug-treated cells.
Cells were harvested at 0, 24, 48, and 72 hours post-treatment. Cell viability was measured by a
luminescent cell viability assay, which measures the ATP generated in viable cells. We found no
significant difference in the viability of control PBMCs and HTLV-1-transformed cells treated with
FTI-277 (Figure 1A). In contrast, the results presented in Figure 1B demonstrate that compared to
control PBMCs, the HTLV-1-transformed cell lines were more sensitive to treatment with GGTI-298
than were control PBMCs (Figure 1B). The viability of C8166 cells was also analyzed by trypan blue
exclusion assays and we found decreased cell viability, similar to that seen using the luminescent assay
(data not shown). The sensitivity to GGTI-298 varied in the HTLV-1-transformed cells with C8166
cells being the most sensitive, followed by SP, C91/PL, and MT2 cells.
To determine the effects of increasing GGTI-298 concentrations on cell viability, final concentrations
of 0, 2.5, 5, and 10 μM of the drug were added to the culture media of HTLV-1-transformed, HTLV-1-
infected cells, and PBMCs for 72 hours. Following treatment, cell viability was determined. As shown
in Figure 1C, the HTLV-1-transformed and infected cells were more sensitive to GGTI-298 and
showed a dose-dependent loss of viability. After 72 hours of treatment with 10 μM GGTI, control
PBMCs showed less than 30% decrease in viability. In contrast, HTLV-1-transformed cells, C8166
and C91/PL, and HTLV-1-infected SP cells showed a 70 to 95% reduction in viability. As above, MT2
cells were the least sensitive of the HTLV-1-transformed cells, with a 40% decrease in viability.

Viruses 2011, 3 1818
Figure 1. GGTI-298 decreases cell viability of Human T-cell leukemia virus type-1
(HTLV-1)-transformed cells. Control PBMCs from three donors and HTLV-1-transformed
SP, C8166, C91/PL, and MT2 cells were treated with (A) 20 μM FTI-277 or (B) 10 μM
GGTI-298. At the indicated times, cells were collected and cell viability determined.
Values for cells treated with FTI-277 or GGTI-298 were compared to the signal for cells
treated with DMSO solvent alone to determine percent cell viability. Error bars represent
the standard error of the mean from three independent experiments performed in triplicate.
(C) Control PBMCs and HTLV-1-transformed SP, C8166, C91/PL, and MT2 cells were
treated for 72 hours with increasing amounts of GGTI-298 and cell viability measured as
before. Error bars represent the standard error of the mean from three independent
experiments performed in triplicate. For all GGTI298-treated cells p < 0.05 compared with
mock-treated control, except MT2 cells treated with 2.5 and 5 μM GGTI-298.

Viruses 2011, 3 1819
2.2. GGTI-298 Induces G2/M Phase Accumulation in HTLV-1-Transformed Cells
GGTI-298 has been shown to inhibit Rho family GTPase activation and reduce cell viability in a
number of cancer cell types, including transformed uninfected T-cells [17,27–35]. Although PBMCs
have a reduced proliferation compared to HTLV-1-transformed cells, GGTI-298 clearly decreased the
viability of HTLV-1-transformed cells. Therefore, we examined its effect on cell cycle progression in
HTLV-1-transformed C8166 and C91/PL cell lines and a Tax-negative cell line, TL-Om1. Cells were
treated with DMSO or 10 μM GGTI-298 for 48 hours and DNA content was analyzed by flow
cytometry after staining with propidium iodide. As shown in Figure 2, GGTI-298 caused accumulation
of C8166 and TL-Om1 cells in the G2/M phase. GGTI-298-treated C91/PL cells showed a small
increase in G2/M phase accumulation in C9/PL cell, indicating that the effect of GGTI-298 on cell
cycle distribution in HTLV-1-transformed cells is cell line dependent. Though both C8166 and
TL-Om1 cells showed a two-fold increase in the percentage of cells in G2/M phase, only TL-Om1 cells
showed an increase in cells in G1 phase following treatment with GGTI-298. In each of the cell lines
treated with GGTI-298, there was a decrease in the percentage of cells in S-phase of the cell cycle.
These results demonstrate that GGTI-298 affects the cell cycle checkpoint pathways by inducing
accumulation of HTLV-1-transformed cells in the G2/M phase of the cell cycle. This is consistent with
other studies that show check point effects in other tumor cell lines [15,29,32,36,37].
Figure 2. GGTI-298 induces G2/M phase accumulation in HTLV-1-transformed cells.
HTLV-1-transformed C8166, C91/PL, and TL-Om1 cells were treated with DMSO or
10 μM GGTI-298. At 48 hours post-treatment, cells were fixed and stained with propidium
iodide. Representative graphs from three independent experiments are shown for untreated
and GGTI-298-treated C8166, C91/PL, and TL-Om1 cells. Percentage of cells in G2/M
phase is shown above each bar.

Viruses 2011, 3 1820
2.3. GGTI-298 Inactivates the NF-
κB Pathway
GGTI-298 has been shown to inactivate AKT and the downstream NF-κB pathway [17,19].
Constitutive activation of NF-κB in HTLV-1-transformed cells plays a key role in cell survival, cell
cycle progression, and prevention of apoptosis in HTLV-1-infected cells. To determine whether
GGTI-298 affected NF-κB activation in HTLV-1-transformed cells, C8166, C91/PL, and TL-Om1
cells were treated with 10 μM GGTI-298 and collected at 0, 8, 24, and 48 hours post-treatment for
immunoblot analysis. Decreased levels of IκBα, the inhibitor of NF-κB were seen by 48 hours
post-treatment in C8166, C91/PL, and TL-Om1 cells (Figure 3A). Likewise, phosphorylated IκB
(p-IκBα) levels decreased similarly to overall IκB levels. Interestingly, p-IκBα levels were initially
low in TL-Om1 cells, peaked at 8 hours post-treatment, and similar to C8166 and C91/PL cells,
decreased by 48 hours post drug treatment (Figure 3A).
To determine whether GGTI-298 affected NF-κB transcriptional activity, real-time PCR analysis
was performed on downstream targets of NF-κB. Cells were treated for 48 hours with 10 μM
GGTI-298 and total RNA was extracted and retrotranscribed. mRNA levels of the NF-κB-regulated
genes iNOS and IL-6 were normalized to 18s RNA levels. A reduction in the levels of both NF-κB-
regulated genes was seen in all three cell lines after treatment with GGTI-298. For C8166 cells, there
was a decrease of 65 to 80% in iNOS and IL-6 mRNA levels following drug treatment. While
treatment of C91/PL and TL-Om1 cells with GGTI-298 showed a decrease of 60% and 40% in iNOS
mRNA levels, IL-6 mRNA levels were less affected with a decrease of about 25%. These data suggest
that the mechanism of reduction in NF-κB-regulated gene expression by GGTI-298 is likely gene and
cell-specific.
Figure 3. GGTI-298 inactivates the NF-κB pathway. (A) C8166, C91/PL, and TL-Om1
cells were treated with 10 μM GGTI-298 and whole cell extracts were prepared at 0, 8, 24,
and 48 hours post-treatment. Immunoblots using antibodies to IκB, p-IκB or actin as
indicated. Normalized densitometric volume is shown above each immunoblot set.
(B) C8166, C91/PL, and TL-Om1 cells were treated with DMSO or 10 μM GGTI-298 and
total RNA was extracted and retrotranscribed at 48 hours post-treatment. cDNA
quantification for 18s and NF-κB targets iNOS and IL-6 was performed by real-time PCR.
mRNA levels were normalized to 18s RNA levels from the same well. Normalized mRNA
levels from cells treated with DMSO solvent alone was set to 1 and mRNA levels
from drug-treated cells were determined relative to this value. Error bars represent the
standard error of the mean from three independent experiments performed in triplicate.
(C) The 4×-NF-κB-Luc luciferase reporter plasmid was cotransfected with RSV-β-
galactosidase reporter plasmid into C8166 cells. Four hours after transfection, cells were
treated with increasing amounts of GGTI-298. Forty-eight hours after treatment with
GGTI-298, cells were harvested and luciferase activities measured. Luciferase activity was
normalized to β-galactosidase activity from the same well. Error bars represent the
standard error of the mean from four independent experiments performed in duplicate.
* p < 0.05; ** p < 0.005 compared with mock-treated control. (D) Effect of GGTI-298 on
NF-κB DNA binding activity was assessed by EMSA using a consensus NF-κB

Viruses 2011, 3 1821
oligonucleotide probe. C8166 cells were treated with 10 μM GGTI-298 and nuclear
extracts were prepared 48 hours post-treatment. For the competition assay, 200-fold molar
excess of unlabeled consensus or mutated oligonucleotide was added to the reaction.

Viruses 2011, 3 1822
The effect of GGTI-298 on NF-κB transcriptional activity was directly analyzed using a 4×NF-κB-
Luc luciferase reporter construct. Four hours post-transfection, C8166 cells were treated with
increasing amounts of GGTI-298 or DMSO solvent. Forty-eight hours post-treatment, cells were
collected and luminescence was measured. Cells treated with 5 μM GGTI-298 showed 40% less
NF-κB transcriptional activity than cells treated with DMSO alone (Figure 3C). Treatment with 10 μM
GGTI-298 reduced NF-κB transcriptional activity by 80%. These results suggest that GGTI-298
inhibits NF-κB transcriptional activity in HTLV-1-infected cells.
To further define the inhibition of NF-κB activity by GGTI-298, nuclear extracts from C8166 cells
treated for 48 hours with 10 μM GGTI-298 were incubated with biotin-labeled NF-κB consensus
sequence oligonucleotide probe and analyzed by electrophoretic mobility shift assay (EMSA).
Consistent with the immunoblot and promoter activation results, GGTI-298 reduced NF-κB complex
formation on its cognate DNA binding site (Figure 3D, lanes 1–2). The specificity of complex
formation was demonstrated by competition assays in which excess unlabeled wild-type, but not
mutant, NF-κB consensus oligonucleotide was able to compete for NF-κB complex formation
(Figure 3D, lanes 3–4). Taken together, these results demonstrate that inhibition of protein
geranylgeranylation inhibits the NF-κB pathway in HTLV-1-transformed cells.
2.4. The Effects of GGTI-298 on HTLV-1-Transformed Cells Are p53-Independent
Previous studies have demonstrated that Tax-mediated activation of a novel NF-κB pathway
functionally inhibits p53 in HTLV-1-transformed cells [10]. NF-κB and AKT inhibitors or IκB
mutants that inhibit NF-κB activation have been reported to reactivate p53 function in Tax-expressing
cells. As GGTI-298 inhibited NF-κB in HTLV-1-transformed cells, we asked whether GGTI-298-
induced G2/M phase accumulation and reduced viability was dependent on p53 reactivation.
To determine whether GGTI-298 affected p53 protein levels, C8166, C91/PL, and TL-Om1 cells
were treated with 10 μM GGTI or 9AA and whole cell extracts collected at 0, 8, 24, and 48 hours
post-treatment for immunoblot analyses. 9AA was included as a control since it has previously been
shown to reactivate p53 function in HTLV-1-transformed cells [14]. We found no increase in p53
protein levels in GGTI-298-treated cells (Figure 4A). Note that the TL-Om1 cell line has a mutant
functionally inactive p53 protein and thus we would not expect p53 to play a role in drug sensitivity in
this cell line.
To directly analyze the effect of GGTI-298 on p53 transcriptional activity, we transfected C8166
cells with a p53-responsive reporter, PG13-Luc, that contains 13 repeats of the p53 response sequence
upstream of the basal promoter driving luciferase. Four hours post-transfection, cells were treated with
increasing amounts of GGTI-298, DMSO solvent alone or 9AA. Forty-eight hours post-treatment,
cells were collected and luminescence measured. We observed a 4-fold increase in p53 transcriptional
activity in cells treated with 9AA compared to cells treated with DMSO solvent alone (Figure 4B). In
contrast, p53 transcriptional activity was not increased in cells treated with GGTI-298 at 2.5 to 10 μM
concentrations, but rather the activity was decreased. This decrease in p53 transcriptional activity is
consistent with the decrease in p53 protein levels (Figure 4A).
Real-time PCR analysis was performed on downstream targets of p53 in cells treated for 48 hours
with 10 μM GGTI-298 (Figure 4C). mRNA levels of the p53-regulated genes MDM2 and PIG3 were

Viruses 2011, 3 1823
normalized to 18s RNA levels. In TL-Om1 cells, which contain a mutant p53, MDM2 and PIG3
mRNA levels increased 65% and 15%. In contrast, MDM2 and PIG3 mRNA levels decreased 50 to
60% in GGTI-298-treated C8166 and C91/PL cells. These results demonstrate that GGTI-298 does not
reactivate p53 in HTLV-1-transformed cells and that the effects of GGTI-298 are p53-independent.
Figure 4. The effects of GGTI-298 on HTLV-1-transformed cells are p53-independent.
(A) C8166, C91/PL, and TL-Om1 cells were treated with 10 μM GGTI-298 or 9AA and
whole cell extracts were prepared at 0, 8, 24, and 48 hours post-treatment. Immunoblots
using antibodies to p53 or actin as indicated. Normalized densitometric volume is shown
above each immunoblot set. (B) PG13-Luc luciferase reporter plasmid was cotransfected
with RSV-β-galactosidase reporter plasmid into C8166 cells. Four hours after transfection,
cells were treated with increasing amounts of GGTI-298 or 10 μM 9AA. Forty-eight hours
after treatment with GGTI-298, cells were harvested and luciferase activities measured.
Luciferase activity was normalized to β-galactosidase activity from the same well. Error
bars represent the standard error of the mean from three independent experiments
performed in duplicate. * p < 0.05; ** p < 0.005 compared with mock-treated control.
(C) C8166, C91/PL, and TL-Om1 cells were treated with DMSO or 10 μM GGTI-298 and
total RNA was extracted and retrotranscribed at 48 hours post-treatment. cDNA
quantification for 18s and p53 targets MDM2 and IL-6 was performed by real-time PCR.
mRNA levels were normalized to 18s RNA levels from the same well. Normalized mRNA
levels from cells treated with DMSO solvent alone was set to 1 and mRNA levels from
drug-treated cells were determined relative to this value. Error bars represent the standard
error of the mean from three independent experiments performed in triplicate.

Viruses 2011, 3 1824
Figure 4. Cont.
2.5. GGTI-298 Inhibits Transcription of the HTLV-1-LTR and Decreases Tax Expression
In addition to regulation of NF-κB, Rho family members have been shown to regulate transcription
of cellular genes through CREB, SRF, and c-Jun transcription factor binding sites, each of which are
present in the Tax-responsive elements of the HTLV-1 promoter and have a role in viral gene
expression [21–25]. Thus we next examined whether GGTI-298 affected transcriptional activation of
the viral promoter. Real-time PCR analysis was performed on tax/rex mRNA levels in C8166 and
C91/PL cells treated for 48 hours with 10 μM GGTI-298 (Figure 5A). As before, target mRNA levels
were normalized to 18s RNA levels. We found a 50% decrease in tax/rex mRNA levels in both C8166
and C91/PL cells following drug treatment. To determine whether GGTI-298 affects Tax protein
expression, C8166 cells were collected for immunoblot analysis at 0, 8, 24, and 48 hours after
treatment with GGTI-298. We found that Tax protein levels decreased approximately 75% after
24 hour treatment with GGTI-298 (Figure 5B). C8166 cells were treated with increasing amounts of
GGTI-298 four hours after being transfected with an HTLV-1-LTR-luciferase construct. Cells were
collected 48 hours post-treatment and luminescence was measured. We found a dose-dependent
repression of viral promoter activity by GGTI-298 (Figure 5C). C8166 cells treated with 10 μM
GGTI-298 showed 95% less HTLV-1 promoter activity as compared to cells treated with DMSO
solvent alone. The decrease in HTLV-1-LTR activity in GGTI-298-treated C8166 cells may be in part
due to diminishment of CRE pathway activity. In a CRE-luciferase reporter assay, we measured a 50%
decrease in luciferase levels in C8166 cells treated with 10 μM GGTI-298 for 48 hours (data not
shown). Together, this data shows that GGTI-298 significantly inhibits transcription of the HTLV-1-
LTR and decreases Tax expression.
Thus, deactivation of NF-κB and accumulation of cells in G2/M phase in GGTI-298-treated HTLV-
1-transformed cells may be, in part, due to decreased Tax expression and activity following inhibition
of transcription pathways by GGTI-298. Indeed, preliminary experiments from another lab revealed
that an HTLV-1-infected human T-cell line could not survive knockdown of Tax protein levels after
being infected with a lentivirus that encoded siRNA targeting Tax [38].

Viruses 2011, 3 1825
Figure 5. GGTI-298 inhibits transcription of the HTLV-1-LTR and decreases Tax
expression. (A) C8166 and C91/PL cells were treated with DMSO or 10 μM GGTI-298
and total RNA was extracted and retrotranscribed at 48 hours post-treatment. cDNA
quantification for tax/rex and 18s was performed by real-time PCR. mRNA levels were
normalized to 18s RNA levels from the same well. Normalized mRNA levels from cells
treated with DMSO solvent alone was set to 1 and mRNA levels from drug-treated cells
were determined relative to this value. Error bars represent the standard error of the mean
from three independent experiments performed in triplicate. (B) C8166 cells were treated
with 10 μM GGTI-298 and whole cell extracts were prepared at 0, 8, 24, and 48 hours
post-treatment. Immunoblots using antibodies to Tax or actin as indicated. Normalized
densitometric volume is shown at top. (C) HTLV-1-LTR-Luc luciferase reporter plasmid
was cotransfected with RSV-β-galactosidase reporter plasmid into C8166 cells. Four hours
after transfection, cells were treated with increasing amounts of GGTI-298. Forty-eight
hours after treatment with GGTI-298, cells were harvested and luciferase activities
measured. Luciferase activity was normalized to β-galactosidase activity from the same
well. Error bars represent the standard error of the mean from three independent
experiments performed in duplicate. ** p < 0.005 compared with mock-treated control. (D)
C8166 cells were infected with adenovirus expressing either GFP (Ad-GFP) or Tax (Ad-
Tax). At 24 hours post-infection, cells were treated with 10 μM GGTI-298. At 48 hours
post-infection, whole cell extracts were prepared and immunoblots were performed using
antibodies to Tax, p-IκB, or actin as indicated. Normalized densitometric volume is shown
above each immunoblot set. (E) The 4×-NF-κB-Luc or (F) HTLV-1-LTR-Luc luciferase
reporter plasmid was cotransfected with RSV-β-galactosidase reporter plasmid into C8166
cells. Twenty-four hours post-transfection, cells were infected with Ad-GFP or Ad-Tax
and treated 4 hours later with 10 μM GGTI-298. Forty-eight hours after treatment with
GGTI-298, cells were harvested and luciferase activities measured. Luciferase activity was
normalized to β-galactosidase activity from the same well. Error bars represent the
standard error of the mean from three independent experiments performed in duplicate.
* p < 0.05; ** p < 0.005 compared with mock-treated control.

Viruses 2011, 3 1826
Figure 5. Cont.
We next determined whether the effect of GGTI-298 on the NF-κB pathway was due to decreased
Tax protein expression in Tax positive cells [28]. C8166 cells were infected with either an adenovirus
construct that expresses Tax (Ad-Tax) or GFP (Ad-GFP). Twenty-four hours post-infection, cells were
treated with 10 μM GGTI-298 and incubated an additional 24 hours. Immunoblots were then
performed and phosphorylation status of IκB analyzed (Figure 5D). As expected from previous
experiments (Figure 3A), GGTI-298 decreased the levels of phosphorylated IκB in cells infected with
Ad-GFP and Ad-Tax as compared to control treated cells. Interestingly, GGTI-298 reduced Tax
protein levels in cells infected with the Ad-Tax construct (Figure 5D, lanes 3–4). Importantly,
however, Tax protein expression was still significantly higher than in mock-treated cells infected with
the Ad-GFP construct (Figure 5D, lane 1). Despite the high level of Tax expression, p-IκB levels still
decreased in response to GGTI-298 treatment (Figure 5D). In addition, increased Tax expression did
not abrogate the effects of GGTI-298 on NF-κB (Figure 5E) or HTLV-1-LTR (Figure 5F)
transcriptional activity. In mock-treated cells expressing Ad-Tax, there was a four-fold increase in
NF-κB transcriptional activity and a two-fold increase in HTLV-1-LTR activation compared to cells
expressing Ad-GFP. Treatment of Ad-Tax and Ad-GFP expressing cells with GGTI-298 significantly

Viruses 2011, 3 1827
inhibited NF-κB transcriptional activity and HTLV-1-LTR activation. While Ad-Tax activity was able
to augment basal Tax-mediated transcriptional transactivation, it was unable to counter the effects of
GGTI-298 on transcriptional repression. Together these results indicate that GGTI-298 decreases Tax
protein levels and HTLV-1-LTR activity and that deactivation of the NF-κB pathway by the inhibitor
does not correlate with Tax expression.
2.6. Discussion
Rho family GTPases regulate multiple signaling pathways that control cytoskeletal organization,
transcription, cell cycle progression and cell proliferation. Overexpression of multiple Rho family
members in human tumors suggests that these proteins are important in tumorigenesis and therefore
potential candidates for therapeutic intervention [39]. Currently, a role for Rho family GTPases in the
development of ATL is emerging. First, Tax has been reported to form complexes with several
members of the Rho family including RhoA, Rac, Gap1m and Cdc42 [40]. Second, microarray
analysis showed altered gene expression of numerous Rho GTPase and GTPase-binding proteins in
ovine B-cells expressing bovine leukemia virus Tax (TaxBLV) [41]. Third, bisphosphonate incardonate,
a compound that disrupts protein farnesylation and geranylgeranylation, has recently been
demonstrated to induce apoptosis in HTLV-1-transformed cells [42]. Fourth, Nonaka et al. reported
that inhibition of geranylgeranylation, but not farnesylation, induced apoptosis in HTLV-1-infected
ATL cell lines [28]. Nonaka et al. further reported that the Rho family GTPases Rab5B and Rac1A
were found to be prenylated in these cells. However, the authors did not define the mechanism used by
GGTI-298 to cause cell death in the HTLV-1-transformed cells.
Here, we show that the prenylation inhibitor GGTI-298 decreased cell viability and induced G2/M
phase accumulation in HTLV-1-transformed cells in a p53-independent manner. In contrast, the
prenylation inhibitor FTI-277 did not affect cell viability in these cells. Treatment of HTLV-1-
transformed cells with GGTI-298 decreased activation of the NF-κB signaling pathway. In addition,
HTLV-1-LTR transcriptional activity was inhibited and Tax protein levels decreased following
treatment with GGTI-298. The results of these experiments suggest a decrease in cellular transcription
whose pattern differs by gene and cell type. However, more pathways need to be examined to
determine specificity. We hypothesize that activated Rho family GTPases have a role in several cell
signaling pathways that are dysregulated in HTLV-1-transformed cells and that geranylgeranyl
transferase inhibitors may provide a novel class of therapeutic agents for treatment of ATL.
GGTI-298 may be useful for the study of other functions of Rho GTPases in HTLV-1-infected
cells such as cell adhesion, migration, and chemotaxis. ATL is characterized by infiltration of
circulating ATL cells into a variety of tissues. Fresh ATL cells from patients express lymphocyte
function-associated antigen 1 (LFA-1) [43]. LFA-1 mediates adhesion of circulating leukocytes to
endothelial ligands and mediates transendothelial migration of ATL cells [44]. Recently, it has been
shown that GGTI-298 is able to diminish LFA-1 expression in T-acute lymphoblastic leukemia cells
by inhibiting the GTPase Rap1b [45]. Interestingly, HTLV-1 p8 increases clustering of LFA-1 on the
cell surface to enhance cell-cell adhesion to promote viral spread [46,47]. By demonstrating here that
HTLV-1-transformed cells are sensitive to GGTI-298, we speculate that this compound could disrupt
HTLV-1 p8-mediated modulation of cell adhesion in these cells by inhibiting LFA-1 clustering.

Viruses 2011, 3 1828
Continued study and characterization of geranylgeranylated proteins, such as Rho family GTPases,
will increase our understanding of the mechanisms that regulate infection and transformation of
HTLV-1-infected cells.
3. Experimental Section
3.1. Preparation of PBMCs
Heparinized human peripheral blood was obtained from healthy volunteers at the NIH
clinical center. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors using
Ficoll-Paque Plus (GE Healthcare, Chalfont St. Giles, UK) according to the manufacturer’s
instructions. Isolated lymphocytes were washed in PBS and activated in the presence of 2 μg/mL
phytohemagglutinin-L (PHA-L) for 24 hours before being cultured in RPMI media supplemented with
20% FBS, 2 mM L-glutamine, penicillin/streptomycin and 50 U/mL interleukin-2 (IL-2).
3.2. Cell Lines and Drug Treatment
The HTLV-1-transformed cell lines C8166, C91/PL, and MT2, the ATL cell line TL-Om1
were maintained in RPMI media supplemented with 10% FBS, 2 mM L-glutamine and
penicillin/streptomycin. IL-2-dependent HTLV-1-infected SP cells were cultured in RPMI media
supplemented with 20% FBS, 2 mM L-glutamine, penicillin/streptomycin and 50 U/mL IL-2. FTI-277
(Sigma, St. Louis, MO, USA), GGTI-298 (Sigma, St. Louis, MO, USA) and 9-aminoacridine (9AA)
(Sigma, St. Louis, MO, USA) were solubilized in dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO,
USA). During drug treatment, mock treated cells were cultured in an equal amount of DMSO (0.1%).
3.3. Whole Cell and Nuclear Extracts Preparation
Approximately 5 × 106 cells were washed in 0.5 volumes of cold 0.01 M PBS and pelleted. For
whole cell extract preparation, cells were incubated in cell lysis buffer (50 mM Tris-HCl, pH 7.4,
120 mM NaCl, 5 mM EDTA, 0.5% NP-40, 0.2 mM Na3VO4, 50 mM NaF) with 1 mM DTT, 1 mM
PMSF, and protease inhibitors on ice for 20 minutes, centrifuged and the supernatant collected. For
nuclear extract preparation, cells were lysed with NE-PER Nuclear and Cytoplasmic Extraction Reagents
according to manufacturer’s instructions (Pierce, Rockford, IL, USA). Protein concentrations were
determined by Bradford assay.
3.4. Immunoblot Analysis
Protein samples of 20 to 50 μg was separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (4–12% or 12%) in MES SDS running buffer (Invitrogen, Carlsbad, CA, USA) and
transferred to Immobilon-P membranes (Millipore, Billerica, MA, USA). The membranes were
blocked with PBS containing 0.1% Tween and 2.5% nonfat dried milk, dried and then probed with the
appropriate primary and horseradish peroxidase-conjugated secondary antibodies. Immunoreactivity
was detected with SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL, USA).
Densitometric volume analyses were performed with AlphaEase FC (Alpha Innotech, Santa Clara, CA,

Viruses 2011, 3 1829
USA) [48]. Protein levels were normalized to actin levels from the same well. Normalized protein
levels were compared with control protein level, which was set to 1, to determine relative
protein levels.
3.5. Antibodies
IκB, p-IκB, (Cell Signaling, Danvers, MA, USA), actin (Sigma, St. Louis, MO, USA), and p53
(Calbiochem, San Diego, CA, USA), primary antibodies and horseradish peroxidase-conjugated
secondary antibodies (GE Healthcare, Chalfont St. Giles, UK) were purchased. Tab172 monoclonal
antibodies were used to detect the expression of Tax protein.
3.6. Cell Viability Assays
Cell viability assays were performed using the CellTiter-Glo luminescent cell viability
assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Briefly,
2.5 × 105 cells/mL were cultured in sterile 96-well plates in the presence of GGTI-298 or DMSO in
RPMI medium. Following incubation, 100 μL of CellTiter-Glo reagent was added to lyse the cells.
The contents were mixed on a rocker for 2 minutes and then incubated at room temperature for
10 minutes. Luminescence was recorded in a luminometer with an integration time of 1 second per
well. The luminescent signals from the GGTI-298-treated cells were normalized to the luminescent
signal from cells treated with DMSO alone, which was arbitrarily set to 100%.
3.7. Flow Cytometry Analysis of Cell Cycle Progression
One to 2 × 106 cells were treated with 10 μM GGTI-298 or DMSO for 48 hours, washed in PBS,
and fixed in 5 mL 70% ethanol overnight at −20 °C. Cells were washed in PBS and stained with
0.5 mL propidium iodide staining buffer (50 μg/mL propidium iodide diluted in PBS with 0.1%
Triton-X-100) for 20 minutes. Samples were acquired on a FACSCalibur flow cytometer using ModFit
LT software (Verity Software House, Topsham, ME, USA) [49] and data was analyzed using FlowJo
software (Tree Star, Ashland, OR, USA) [50].
3.8. Transfection and Luciferase Assays
Three to 4 × 106 cells were transfected with 1 μg RSV-β-galactosidase and 4 μg 4×-NF-κB-Luc,
PG13-Luc, or HTLV-1-LTR-Luc luciferase reporter plasmids by nucleofection with Human T-cell
Nucleofector Kit/nucleofection program U-014 (Amaxa, Gaithersburg, MD, USA) according to
manufacturer’s instructions. All plasmids were previously described [13]. Immediately after
nucleofection, 5 × 105 cells/well were distributed on 24 well-plates. Four hours after nucleofection,
cells were treated with GGTI-298, 9AA or DMSO. Forty-eight hours after treatment, cells were lysed
in passive lysis buffer and luciferase activity was measured using the dual luciferase reporter assay
system (Promega, Madison, WI, USA) and Galactolight assay kit (Tropix, Bedford, MA, USA).
Luminescence was recorded in a luminometer with an integration time of 12 seconds per sample.
Luciferase activity was normalized to β-galactosidase activity from the same well. Normalized

Viruses 2011, 3 1830
luciferase activity from cells treated with DMSO solvent alone was set to 1 and luciferase values from
each drug concentration were determined relative to this value.
3.9. Real-Time PCR
For real-time PCR analysis, 1 × 106 cells were treated with 10 μM GGTI-298 or DMSO for
48 hours. Cells were washed in PBS and total RNA was extracted from all samples with RNeasy Plus
(Qiagen, Valencia, CA, USA) and retrotranscribed with Quantitect Reverse Transcription kit (Qiagen,
Valencia, CA, USA), according to manufacturer’s instructions. The cDNA quantification for 18s, IL6,
iNOS, MDM, PIG3 and tax/rex was performed by real-time PCR using an ABI 7000. Reactions were
performed using a SYBR FAST PCR mix (Kapa Biosystems, Woburn, MA, USA), following the
manufacturer’s instructions. Primer sequences were designed to specifically amplify:
18s, 5'-GCCCGAAGCGTTTACTTTGA-3' (forward) and
5'-TCCATTATTCCTAGCTGCGGTATC-3' (reverse);
IL6, 5'-GGTACATCCTCGACGGCATC-3' (forward) and
5'-CCAGTGCCTCTTTGCTGCTT-3' (reverse);
iNOS, 5'-CAAGGCATCCTGGAGCGAGT-3' (forward) and
5'-GTAGGTGAGGGCCTGGCTGA-3' (reverse);
MDM, 5'-GGTTGACTCAGCTTTTCCTCTTG-3' (forward) and
5'-GGAAAATGCATGGTTTAAATAGCC-3' (reverse); PIG3, 5'-CACTCCCAACGGCTCCTTT-3'
(forward) and 5'-GCCCATCTTGAGCATGGGTG-3' (reverse); tax/rex primers were previously
described [51]. Results were expressed as ΔΔCt, where Ct is the cycle threshold, and presented as
ratios between the target gene and the 18s housekeeping RNA. Normalized mRNA levels from cells
treated with DMSO solvent alone was set to 1 and mRNA levels from drug-treated cells were
determined relative to this value.
3.10. Electrophoretic Mobility Shift Assays (EMSAs)
The sequence used for NF-κB consensus wild-type oligonucleotide was
5'-AGTTGAGGGGACTTTCCCAGGC-3', while the NF-κB mutant oligonucleotide was
5'-AGTTGAGGCGACTTTCCCAGGC-3'. Double-stranded NF-κB consensus wild-type oligonucleotide
was labeled with biotin-11-dUTP (Pierce, Rockford, IL, USA). EMSA reactions included 10× EMSA
buffer (100 mM Tris pH7.5, 500 mM KCl, 10 mM DTT, 2.5% glycerol, 5 mM MgCl, 50 ng
poly(dI/dC), 2.5 μg nuclear extract and approximately 20 fmol labeled probe in a 20 μL total reaction
volume. For competition assays, approximately 200-fold molar excess of unlabeled oligonucleotide
was added to the reaction. Reactions were incubate6+d at room temperature for
20 minutes. Complexes were resolved on a 5% nondenaturing polyacrylamide gel in 0.5× TBE.
Following electrophoresis, binding reactions were transferred to a Biodyne B membrane (Pierce,
Rockford, IL, USA) and complexes were detected by streptavidin-horseradish peroxidase
chemiluminescent substrate.

Viruses 2011, 3 1831
3.11. Adenovirus Infection of Cells
An adenovirus-Tax (Ad-Tax) construct provided by M. Yoshida and an adenovirus-green
fluorescent protein (Ad-GFP) construct (Q-Biogene, Montréal, Canada) were used to infect
HTLV-1-transformed C8166 cells (1 × 106 cells) in RPMI media not supplemented with serum.
The adenovirus constructs were previously described [52]. At three hours post-infection, cells were
washed and resuspended in RPMI media supplemented with 10% FBS, 2 mM L-glutamine and
penicillin/streptomycin. The infected cells were incubated for 24 hours for GFP and Tax expression
and then treated with 10 μM GGTI-298 for an additional 24 hours.
3.12. Statistical Analysis
Data were analyzed by Student’s two-sample equal variance t test with a p value of <0.05
considered significant.
4. Conclusions
In conclusion, we have demonstrated that treatment of HTLV-1-transformed cells with GGTI-298,
a prenylation inhibitor that inhibits geranylgeranylation, decreased cell viability and induced G2/M
phase accumulation. The effects of GGTI-298 observed in HTLV-1-transformed cells suggest an
important role for geranylgeranylated proteins in cell survival and cell cycle progression. Continued
study and characterization of geranylgeranylated proteins, such as Rho family GTPases, will increase
our understanding of the mechanisms that regulate transformation of HTLV-1-infected cells.
Acknowledgements
This research was supported by the Intramural Research Program of the National Institute of Health,
National Cancer Institute, Center for Cancer Research. We would like to thank the Brady lab for
thoughtful discussion.
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