From the Medical Scientist Training Program, the
¶ Cell and Molecular Biology Graduate Program, and the
** Department of Microbiology and Immunology and the Comprehensive
Cancer and Geriatrics Center, University of Michigan Medical Center
CCGC 3217, Ann Arbor, Michigan 48109-0934
Received for publication, March 1, 2001, and in revised form, April 17, 2001
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ABSTRACT |
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Telomerase is a multi-subunit
ribonucleoprotein holoenzyme that stabilizes telomere length through
the addition of new repeat sequence to the ends of chromosomes.
Telomerase reverse transcriptase is the subunit of this complex
responsible for the enzymatic activity of telomerase. Expression of the
reverse transcriptase is regulated at the level of transcription
through the action of transcription factors that target its promoter.
Most Kaposi's sarcoma tumor cells are latently infected with the
Kaposi's sarcoma-associated herpesvirus, and the constitutive
expression of a viral-encoded latency-associated nuclear antigen has
been shown to be important for the maintenance of the viral episome.
The proliferative nature of Kaposi's sarcoma suggests that this
antigen may also play a critical role in viral-mediated oncogenesis. In
this study telomerase reverse transcriptase promoter elements cloned
into a luciferase reporter plasmid were analyzed to determine the
ability of the latency-associated nuclear antigen to regulate
transcription. The latency-associated nuclear antigen transactivated
the full-length promoter in 293T, 293, and BJAB cell lines.
Furthermore, truncation promoter studies implicated sequence from Telomeric DNA, found at the distal ends of chromosomes, consists
of a 6-base pair repeat of the sequence TTAGGG (1). Telomeres have an
average length of 5-15 kilobases and function to prevent chromosome degradation, end-to-end chromosome fusions, and chromosome loss (2, 3). Additionally, telomeres can induce cellular senescence
when they are critically shortened (4, 5). The telomeres of most human
somatic cells shorten with each cycle of chromosome replication because
DNA polymerase is unable to completely replicate the lagging strand
(6). Telomerase is a ribonucleoprotein enzyme complex that functions to
stabilize telomere length via the addition of new repeat sequence (2). Telomerase is active in cells with high regenerative capacity such as
lymphocytes, hematopoietic progenitor cells, keratinocytes, and uterine
endometrial cells (7-9). Moreover, telomerase is typically active in
tumor-derived cell lines as well as in malignant tissues (10-12). The
activation of telomerase is a common and perhaps necessary step as
cells move beyond replicative crisis to immortality associated with
cancer cells (11).
Three subunits of telomerase have been identified. Two components are
expressed in virtually all human cells with no correlation to
telomerase activity. The first is an RNA component, hTR, that provides the template from which additional repeat sequence is constructed (13, 14). The second is an integral protein component, TP1, the human homologue of the Tetrahymena telomerase P80 gene (15, 16). Neither is down-regulated during cellular differentiation when decreases in telomerase activity are observed (15, 17-19). The
third component, hTERT,1 is
responsible for the enzymatic activity of telomerase (20, 21). In
contrast to hTR and TP1, the expression of hTERT correlates with
telomerase activity, and hTERT mRNA is down-regulated during cell
differentiation and up-regulated during cell immortalization (9, 10,
22, 23). The expression of hTERT is likely regulated through the action
of transcription factors targeting the hTERT promoter. An active hTERT
promoter is observed in telomerase-positive cell lines and cancer
cells, whereas the promoter is repressed in telomerase-negative
cells (24, 25).
The products of known oncogenes and tumor suppressors have been shown
to activate and repress, respectively, the hTERT promoter. Following
the initial identification of Myc as an activator (25-27), further
effects were observed in studies with the transactivator Sp1 and with
the repressors WT1, Mad1, p53, and MZF-2, all shown to modulate the
activity of the hTERT promoter (28-32). Although the E6 gene product
of human papillomavirus type 16 has been shown to activate telomerase
by functional assays (33), to date no tumor virus gene product has been
shown to specifically activate the hTERT promoter.
Prior to cases related to the AIDS epidemic of the early 1980s, KS was
a relatively rare skin neoplasm typically afflicting elderly men of
Mediterranean descent, immunosuppressed individuals such as transplant
recipients, and young men of sub-Saharan African countries (34, 35).
The high prevalence of KS among HIV-infected homosexual men and the low
prevalence among HIV-infected hemophiliacs and intravenous drug users
strongly implicated transmission of an infectious agent through sexual
activity (36, 37). KSHV, also referred to as human herpesvirus 8, was
subsequently identified by polymerase chain reaction-based studies and
designated a LANA is a highly immunogenic protein encoded by open reading frame 73 of the KSHV genome (51). The 1162-amino acid LANA protein has definable
domains that include an amino-terminal proline-rich region, an
~100-amino acid acidic domain, a large glutamine-rich repetitive
domain, and a leucine zipper motif (see Fig. 1; Refs. 39 and 52).
Additionally, LANA possesses potential nuclear localization signals and
numerous phosphorylation sites recognized by several common kinases
(53, 54). LANA is constitutively expressed during viral latency and is
important for maintenance of the viral episome during chromosome
replication (55, 56). The proliferative nature of KS and other
KSHV-associated diseases as well as structural motifs of LANA that
potentially interact with a variety of cellular factors suggest a role
for LANA in mediating viral oncogenesis through transcriptional
regulation. Recently, it was shown that LANA antagonizes the tumor
suppressor p53, thereby protecting against cell death (57). LANA has
also been shown to target the retinoblastoma protein regulating E2F responsive promoters (58). Here, we show that LANA likely contributes to cell immortalization by transactivation of the hTERT promoter.
Plasmids, Cell Lines, and Culture Conditions--
The
preparation of pGL3B-hTERT luciferase reporter constructs and the
pA3M-LANA expression construct have been described previously (25, 56).
The pGL3B-hTERT constructs were obtained from J. Carl Barrett. HEK
293 cells are human embryonic kidney cells transformed by adenovirus
type 5 DNA; the HEK 293T cell line is an HEK 293-derived line that
stably expresses the SV40 T-antigen (59). BJAB is a B cell derived from
a patient with Epstein-Barr virus-negative African Burkitt's lymphoma
(60). BJAB and Rat-1 cell lines were obtained from Elliott Kieff.
SUSM-1 cells maintain their telomeres by a telomerase-independent
mechanism and are consequently negative for telomerase activity (61).
The SUSM-1 cell line was prepared by mutagen treatment of fetal human
diploid fibroblasts (62). SUSM-1 cells were provided by Masayoshi
Namba. BC-3 is a KSHV-positive body cavity-based lymphoma-derived cell
line obtained from the American Type Culture Collection (63). HEK 293, HEK 293T, Rat-1, and SUSM-1 cells were grown in Dulbecco's modified
Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal
bovine serum, 2 mM glutamine, 25 units/ml penicillin, 25 µg/ml streptomycin, and 10 µg/ml gentamicin. BJAB cells were grown
in RPMI medium 1640 (Life Technologies, Inc.) supplemented as for
Dulbecco's modified Eagle's medium. BC-3 cells were grown in RPMI
medium 1640 supplemented as for Dulbecco's modified Eagle's medium
but with 20% fetal bovine serum. Cells were grown at 37 °C in a
humidified environment supplemented with 5% CO2.
Transfection and Luciferase Assay--
HEK 293, HEK 293T, Rat-1,
and SUSM-1 cells were collected at 70% confluency by trypsinization
with trypsin-EDTA (Life Technologies, Inc.). 10 million cells were
resuspended, along with plasmid DNA (generally 5 µg of pGL3-Basic
reporter and 10 µg of pA3M expression construct), in 400 µl of
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum. The cells were transfected by electroporation with the
Bio-Rad Gene Pulser II at 210 V and 975 microfarads. Transfected cells
were transferred to 100-mm plates in 10 ml of Dulbecco's modified
Eagle's medium with 10% fetal bovine serum. The plates were incubated
at 37 °C in a humidified environment supplemented with 5%
CO2 for 20 h. BJAB cells were collected at 5 × 105 cells/ml. 10 million cells were transfected at 220 V
and 975 microfarads.
At 20 h, cells were harvested and washed once with
phosphate-buffered saline (Life Technologies, Inc.). Luciferase
activity was determined as per manufacturer's instructions using the
luciferase assay system with reporter lysis buffer (Promega). Briefly,
200 µl of lysates were prepared by freeze/thaw in reporter lysis
buffer. The lysates were subsequently diluted 1:10 in reporter lysis
buffer, and 40 µl of the dilution was mixed with 100 µl of
luciferase assay reagent. Luminescence was measured for 10 s by
the Opticomp I luminometer (MGM Instruments, Inc.). It should be
emphasized that lysates were always diluted to ensure that luciferase
activity was within the linear range of the assay. The results shown
represent experiments performed in triplicate. The experiments were
repeated multiple times to ensure that the observed trends were reproducible.
Western Blot Analysis--
The same lysates prepared for
luciferase assay were aliquoted and mixed 1:1 with 2× SDS gel-loading
buffer (100 mM Tris·Cl, pH 6.8, 200 mM
dithiothreitol, 4% SDS, 0.2% bromphenol blue, and 20% glycerol),
placed at 95 °C for 5 min, and spun to remove cell debris. Soluble
proteins were fractionated by electrophoresis on a 6%
SDS-polyacrylamide gel. The fractionated protein was then transferred
to a 0.45-µm nitrocellulose membrane. The membrane was blocked with
5% dry milk in PBS and was subsequently blotted with human serum,
adsorbed with B cell extracts, and previously shown to be reactive to
LANA (56, 64) at a 1:50 dilution followed by horseradish
peroxidase-linked protein A (Amersham Pharmacia Biotech) at a 1:5000
dilution. The blot was visualized by a standard chemiluminescence
detection protocol (Amersham Pharmacia Biotech).
Immunofluorescence--
BJAB cells were transfected with either
pA3M or pA3M-LANA and incubated as described above. Cells were
harvested and washed with PBS. Slides of transfected BJAB and
nontransfected BC-3 were prepared and fixed with 1:1 acetone:methanol.
The slides were incubated with 20% goat serum for 30 min at room
temperature in a humidity chamber. After washing with PBS, slides were
incubated with specific antibody, either human serum reactive to LANA
at 1:100 dilution or Myc mouse monoclonal IgG at 1:500 dilution, for
2 h. After washing thoroughly with PBS, slides were incubated with
either FITC-conjugated goat anti-human antibody or FITC-conjugated goat
anti-mouse antibody at 1:1000 dilution for 1 h. Slides were washed
thoroughly with PBS, and proteins were visualized on an Olympus BX60
fluorescent microscope. The photographs were captured using an Olympus
digital camera and the Esprit program version 1.2.
In Vitro DNA Binding--
Sp1 DNA probes were prepared by
annealing complementary oligonucleotides containing the GC-rich Sp1 DNA
binding site (Life Technologies, Inc. custom primers). The wild-type
probe sequence was taken from LANA Activates Transcription of the Full-length hTERT Promoter in
HEK 293 Cells--
The proliferative nature of cells latently infected
with KSHV as well as structural domains of LANA having the potential to mediate protein-protein and protein-DNA interactions suggest a role for
LANA in regulating cellular gene expression (Fig.
1). Because expression of hTERT is
primarily regulated at the transcriptional level, the effect of LANA on
hTERT expression was investigated by testing the ability of LANA to
regulate expression from luciferase reporter constructs containing the
hTERT promoter in HEK 293 cells. The HEK 293 cell line has been
employed in previous studies of KSHV infectivity and propagation. In
one study KSHV was cytotoxic to 293 cells and was detected by
polymerase chain reaction in infected cells but not uninfected ones
during serial passage (50). In another study 293 cells were susceptible
to KSHV infection by detection of a spliced late mRNA (49). These
data support the use of the HEK 293 cell line for preliminary
examination of potential LANA effects on gene expression. The region
The hTERT Promoter Element Is Endogenously Activated in Human Cell
Lines--
To assess the possibility that the above response was due
to either the total amount of DNA transfected or the particular cell
line employed, similar assays were performed in five cell lines, HEK
293 and 293T, BJAB, Rat-1 fibroblasts, and SUSM-1 fibroblasts. HEK 293, HEK 293T, BJAB, and Rat-1 fibroblasts have significant endogenous
telomerase activity. In contrast, SUSM-1 fibroblasts maintain
their telomeres by a telomerase-independent mechanism and were
previously shown to have little or no telomerase activity (61). In HEK
293T and 293 cell lines, a 15-fold increase in luciferase activity was
observed when the telomerase promoter was cloned upstream of the
luciferase gene. Similarly, the BJAB cell line showed a 20-fold
increase in activation (Fig.
3A). It should be noted that
pGL3-Basic background activity differed in these three cell lines and
that fold activation of the hTERT promoter was always calculated
relative to the appropriate pGL3-Basic/pA3M vector alone control.
To demonstrate that expression of LANA further augments transcription
at the hTERT promoter, the aforementioned cell lines were transfected
with pGL3B-TRTP luciferase reporter and either LANA expression plasmid
or vector control. In HEK 293T and BJAB cell lines, expression of LANA
consistently resulted in an additional 1.5-2-fold activation of the
hTERT promoter over background activity. Moreover, in HEK 293 cells
expression of LANA resulted in 5-7-fold activation relative to
background (Fig. 3B). These data indicate that LANA can
activate the hTERT promoter over endogenous hTERT promoter activity in
these telomerase-positive cell lines and that this effect is not due to
the total amount of DNA transfected. As discussed in more detail below,
the modest activation observed in HEK 293T and BJAB cell lines was
expected because these lines express potential activators of the hTERT
promoter, SV40 T-antigen and Myc, respectively. Just as expression of
the T-antigen overwhelms LANA-mediated activation of the hTERT promoter
in the HEK 293 background, Myc expression likely limits potential fold
activation of the hTERT promoter in the BJAB cell line.
In comparison, Rat-1 fibroblasts and the telomerase-negative fibroblast
cell line SUSM-1 were examined for LANA-mediated activation of the
hTERT promoter. No significant activation over endogenous hTERT
promoter activity was observed in either fibroblast cell line (Fig.
3B). It should be noted that although SUSM-1 demonstrated less endogenous hTERT promoter activity than the other cell lines examined (Fig. 3A), in our experiments the activation seen
was greater than expected based on previous reports (25, 61). Additionally, it was observed that as the SUSM-1 cell line was expanded
in culture for the transfection experiment, the cells acquired a more
quickly growing phenotype. This change in phenotype is perhaps
attributable to changes in cellular gene expression that partially
activated the hTERT promoter explaining the significant endogenous
activation observed by luciferase assay.
Transiently Transfected LANA Is Expressed in Human Cell
Lines--
To demonstrate that the LANA expression vector employed
here, pA3M-LANA, effectively expresses LANA protein in our transient reporter assay, cell lysates from a number of cell lines were examined
by immunoblot and immunofluorescence. The results demonstrated a series
of bands above the 215-kDa marker in pA3M-LANA-transfected HEK 293T and
293 cells; no bands were present in pA3M control lanes (Fig.
4A, lanes 2 and
4 and lanes 1 and 3, respectively). Protein corresponding to 1.5 million HEK 293 cells was necessary to
mimic the LANA intensity of 0.5 million HEK 293T cells, indicating that
LANA expression was enhanced in HEK 293T relative to HEK 293, probably
because of the presence of the SV40 T-antigen (Fig. 4A,
lanes 2 and 4).
To compare the level of expression in transiently transfected cells
with KSHV-infected cells, immunofluorescence analysis was performed.
Aliquots of BJAB cells were transfected as for hTERT luciferase assays,
and slides were prepared for both transfected BJAB and BC-3, a
KSHV-positive body cavity-based lymphoma-derived cell line (63). In
BC-3 and LANA-transfected cells, FITC-mediated fluorescence was
localized to the nucleus (Fig. 4B, compare left and right panels). To demonstrate that this fluorescence was
specific for the Myc-tagged LANA expressed here, Myc monoclonal IgG was used for both pA3M and pA3M-LANA-transfected BJAB. Nuclear localization of the fluorescence signal was observed for pA3M-LANA-transfected BJAB
but not for the pA3M-negative control (Fig. 4C, compare
left and right panels).
LANA Activates Transcription of Truncated hTERT Promoter
Constructs--
Since the recent cloning of the hTERT promoter, the
products of known oncogenes and tumor suppressors have been shown to
both activate and repress the hTERT promoter via specific protein-DNA interactions (25, 28-32). To examine which region or regions of the
hTERT promoter are responsible for LANA-mediated activation, reporter
plasmids, with serial truncations of the hTERT promoter cloned upstream
of the luciferase gene (25), were cotransfected with LANA expression
plasmid in HEK 293 cells (Fig.
5A). In all cases, both
endogenous activity and LANA-enhanced activity were normalized as fold
activation relative to pGL3-Basic/pA3M vector control as shown in the
left column in panels B-F of Fig. 5. The endogenous or LANA-independent activation of these hTERT truncations increased significantly with the removal of 5' sequence to position LANA Targets the Sp1-DNA Interaction in the Context of the hTERT
Promoter--
The Latent infection with KSHV is believed to play a causal role in
several proliferative lesions, namely KS, body cavity-based lymphoma,
and multicentric Castleman's disease (40, 44-46). KSHV also
transforms primary human endothelial cells in vitro, and by
telomeric repeat amplification protocol assay it was demonstrated that
these KSHV-transformed cells had enhanced telomerase activity; telomerase activity was not detected in uninfected cells (43). Although
the E6 gene product of human papillomavirus type 16 activates telomerase by telomeric repeat amplification protocol assay (33), the
effect of other viruses and their gene products on the regulation of
telomerase activity remains relatively unexplored. Here we propose that
LANA, a constitutively expressed protein detected in latent KSHV
infection, potentially contributes to primary cell transformation and
the proliferative nature of KSHV-infected lesions by activating the
hTERT promoter.
Just as KSHV does not mediate transformation of all cell types
infected, LANA-mediated activation of the hTERT promoter was not
equally efficacious in all cell lines examined here. The most dramatic
activation was observed in the HEK 293 cell line as LANA activated the
wild-type hTERT promoter 5-7-fold. In contrast, activation was
~2-fold in HEK 293T and BJAB cell lines. This discrepancy was
expected and can likely be attributed to differences in endogenous Myc
expression and subsequent activation of the hTERT promoter in these
cell lines. BJAB is a B cell line derived from a patient with
Epstein-Barr virus-negative African Burkitt's lymphoma (60). African
Burkitt's lymphoma is notable for translocation of the Myc gene into
the immunoglobulin heavy chain locus resulting in constitutive high
level Myc expression (67). Myc expression is known to strongly activate
the hTERT promoter (25). Consequently, in the BJAB cell line,
LANA-mediated fold activation was likely less dramatic because of this
already strong endogenous activation mediated by Myc. Similarly, HEK
293T expresses the SV40 T-antigen, a promiscuous transactivator that
has been shown to activate the human Myc promoter (68). In fact,
endogenous activation of the hTERT promoter was 10-fold higher in HEK
293T relative to HEK 293 by transient transfection luciferase assay,
suggesting that endogenous hTERT promoter activity was indeed
up-regulated in the HEK 293T cell line (data not shown). Although the
cell lines employed here provide a good initial screen of LANA-mediated
hTERT expression effects, we are currently planning further experiments to examine these effects in endothelial cells and primary B cells, cell
lines particularly relevant to latent KSHV infection.
Ideally, the cell lines employed in these studies would have
undetectable telomerase activity and, consequently, minimal endogenous activation of the hTERT promoter; however, because up-regulation of
telomerase activity is a common step in cell immortalization, most cell
lines amenable to the assays utilized here have significant detectable
telomerase activity. An exception is the SUSM-1 cell line. This line
was derived by mutagen treatment of fetal human diploid fibroblasts and
maintains telomere length by a telomerase-independent mechanism (61).
However, transient transfection experiments performed to assay for
LANA-mediated hTERT promoter activation indicated that neither SUSM-1
nor Rat-1 fibroblast cell lines facilitated activation. This suggests
that cell-specific factors in the human embryonic kidney and BJAB cell
lines necessary for LANA-mediated activation were absent or sequestered
in these fibroblast cell lines. As was mentioned previously, all cell
lines examined, including the telomerase-negative cell line SUSM-1
(61), showed significant endogenous activation of the hTERT promoter
(Fig. 3A). It would be of interest to further examine
LANA-mediated activation of the hTERT promoter in a human embryonic or
B cell line lacking endogenous hTERT promoter activity.
Promoter truncation studies implicated the 130
to +5 in viral-mediated activation. This region contains five Sp1
transcription factor-binding sites. Electrophoretic mobility shift
assays indicated that the latency-associated nuclear antigen targets
and affects the Sp1-DNA complex in the context of BJAB nuclear extracts.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-herpesvirus placing it in the same family as
the primate Rhadinovirus herpesvirus saimiri and
the human Lymphocryptovirus Epstein-Barr virus (38, 39).
KSHV is strongly linked to Kaposi's sarcoma with infected individuals
converting to seropositivity prior to expressing a disease phenotype
(40). There is a greater than 90% correlation between virus nucleic
acid detection and disease (41). KSHV also targets the
endothelial-derived spindle cell, which is the primary tumor cell
associated with KS (42, 43). KSHV has since been associated with
other malignancies including multifocal Castleman's disease and a rare
B cell lymphoma, the body cavity-based lymphoma or primary effusion
lymphoma (44-46). Body cavity-based lymphoma-derived cell lines are
the only KSHV-infected cells easily amenable to study in culture, and
KSHV infection in these cell lines is predominantly latent (47). More
recently, human-derived microvascular endothelial cells have been shown
to support latent infection with KSHV. These cells undergo
morphological changes resulting in a phenotype that is similar to that
seen in the formation of spindle cells in KS (48). Additionally, KSHV
has been shown to infect a variety of cultured cells, including HEK
293, by reverse transcription-polymerase chain reaction detection of a
spliced late mRNA (49). KSHV has also been detected by polymerase
chain reaction in infected HEK 293 cells but not uninfected ones during
serial passage (50).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
119 to
98 of the hTERT promoter
(GCGCGGACCCCGCCCCGTCCCG). The mutant probe sequence was
GCGCGGACCCCGAACCGTCCCG. Probes were end-labeled with
terminal transferase and [
-32P]dGTP. Sp1 mouse
monoclonal IgG was purchased from Santa Cruz Biotechnology. In
vitro translated LANA was made with the TNT quick-coupled in
vitro translation kit (Promega) according to the manufacturer's
recommendations. Nonspecific competitor DNA was TLBR1, an unrelated
31-base pair probe. BJAB nuclear extract was prepared as previously
detailed (65). Electrophoretic mobility shift assay reactions were
performed as described previously (66).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1665 to +5 of the hTERT promoter was cloned into the pGL3-Basic
luciferase reporter construct such that initiation of transcription via
the hTERT promoter would drive the transcription and ultimately the translation of the luciferase gene (25). This reporter, pGL3B-TRTP, was
transfected into HEK 293 cells along with pA3M-LANA, a Myc-tagged LANA
expression vector. Initial cotransfection of LANA expression vector
with the hTERT reporter construct consistently resulted in 4.5-fold
activation relative to reporter construct alone. Increasing the
concentration of the LANA expression construct consistently augmented
the activation to 7-fold. These data indicate that the transactivation
of the hTERT promoter in the HEK 293 cell line is directly proportional
to the quantity of LANA expressed as demonstrated by the observed
dose-response relationship (Fig. 2).
Further increasing the amounts of LANA resulted in abrogation of this
activity and increased cell death (data not shown).
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Fig. 1.
Schematic representation of the KSHV LANA
polypeptide showing its structural motifs. The proline-rich
domains are located in the amino terminus, and the large central
glutamine/glutamate-rich region is flanked by an acidic domain and a
putative leucine zipper (39, 52). LANA possesses putative nuclear
localization signals (NLS) at both the amino and carboxyl
termini (53, 54). Potential phosphorylation, glycosylation,
myristoylation, and amidation sites are also present but are too
numerous to be displayed here.
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Fig. 2.
LANA transactivates the full-length hTERT
promoter with a dose-response relationship. 10 million human
embryonic kidney 293 cells were transfected with pGL3B-TRTP luciferase
reporter construct and increasing amounts of pA3M-LANA expression
construct as indicated. The promoter activity was expressed as the fold
activation relative to the pGL3B-TRTP alone control. The means and
standard deviations from three independent transfections are
shown.
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Fig. 3.
LANA transactivates the full-length hTERT
promoter relative to vector control in 293T, 293, and BJAB cell
lines. A, 10 million HEK 293T, HEK 293, BJAB, Rat-1, or
SUSM-1 cells were transfected either with 5 µg of pGL3B-TRTP or with
pGL3-Basic control and 10 µg of pA3M vector control. The promoter
activity for each combination of plasmids was expressed as the fold
activation relative to pGL3-Basic/pA3M control. The means and standard
deviations from three independent transfections are shown.
B, cell lines as above were transfected with 5 µg of
pGL3B-TRTP and 10 µg of either pA3M-LANA or pA3M vector control. The
promoter activity for each combination of plasmids was expressed as the
fold activation relative to pGL3B-TRTP/pA3M control. The means and
standard deviations from three independent transfections are
shown.
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Fig. 4.
LANA is expressed in HEK 293T, HEK 293, and
BJAB cells. A, from Fig. 3, selected lysates were
fractionated by electrophoresis on a 6% SDS-polyacrylamide gel.
Volumes corresponding to 0.5 million HEK 293T cells and 1.5 million HEK
293 cells were used. The protein was transferred to a nitrocellulose
membrane; the membrane was blotted with human serum reactive to LANA
from a KS patient followed by horseradish peroxidase-linked protein A. The blot was visualized by a standard chemiluminescence detection
protocol. B, BJAB transfected with pA3M-LANA and
KSHV-infected BC-3 were fixed in 1:1 methanol:acetone and incubated
with KS patient serum reactive to LANA. The secondary antibody used for
detection was FITC-conjugated goat anti-human antibody. C,
BJAB transfected with either pA3M-LANA or pA3M were fixed in 1:1
methanol:acetone and incubated with monoclonal mouse anti-Myc antibody
to detect Myc-tagged LANA. The secondary antibody used for
detection was FITC-conjugated mouse anti-human antibody.
408, perhaps attributable to elimination of repressor binding (Fig.
5, compare B and C, middle columns).
Subsequent removal of 5' sequence to
149 depressed endogenous
activation ~2-fold (Fig. 5, compare D and E,
middle columns). This is consistent with previous reports
that the elimination of the region from
208 to
149 and specifically
the E-box that it contains depresses endogenous hTERT activation (25).
Despite variations in the endogenous activation of the hTERT promoter
with serial 5' deletion, LANA consistently activated transcription
relative to background activity in all constructs examined. To
demonstrate that LANA does not similarly activate transcription of the
luciferase gene in the context of a pGL3-Basic vector with no hTERT
promoter sequence inserted, HEK 293 cells were transfected with
pGL3-Basic and either LANA expression plasmid or vector as was done for
pGL3B-hTERT promoter constructs. Fold activation in the presence of
LANA was then calculated relative to endogenous activation of the
appropriate pGL3 reporter (Fig. 6). These
data demonstrate two important effects. First, although endogenous or
LANA-independent activation of the hTERT promoter differs for each
hTERT promoter truncation (Fig. 5), LANA constitutively activates
transcription ~3-4-fold relative to endogenous promoter activity for
all hTERT promoter truncation constructs tested (Fig. 6). Second, LANA
does not activate transcription of the luciferase gene in the context
of a pGL3-Basic vector with no hTERT sequence inserted (Fig. 6). These
two observations suggest that the smallest hTERT promoter truncation
examined, the
130 to +5 fragment, is sufficient to mediate LANA
activation of the hTERT promoter relative to endogenous activation.
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Fig. 5.
LANA transactivates hTERT promoter
truncations in HEK 293 cells. A, this schematic
demonstrates the full-length hTERT promoter and truncations. Some known
activators (white symbols) and repressors (black
symbols) of the hTERT promoter are indicated. B-F, 10 million HEK 293 cells were transfected with 5 µg of either pGL3-Basic
or hTERT luciferase reporter construct as indicated and 10 µg of
either pA3M vector control or pA3M-LANA as indicated. The promoter
activity for each combination of plasmids was expressed as the fold
activation relative to pGL3-Basic/pA3M control. The means and standard
deviations from three independent transfections are shown.
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Fig. 6.
The specificity of LANA activation of the
hTERT promoter is conferred by an element or elements between 130 and
+5. The fold activation was calculated with the data presented for
HEK 293 cells in Fig. 5. The luciferase activity for cells transfected
with 5 µg of luciferase reporter and 10 µg of pA3M-LANA was divided
by the luciferase activity for cells transfected with 5 µg of the
same luciferase reporter and 10 µg of pA3M vector control. The
error bars represent the summation of relative error for
each of the above values.
130 to +5 region of the hTERT promoter is
significant in that it contains five GC-rich boxes (25). Previous
electrophoretic mobility shift assay experiments have demonstrated
binding of transcription factor Sp1 to these five sites (24); further, it has been suggested that Myc, via the aforementioned E-box, and Sp1
are the major determinants of endogenous hTERT expression (28). To ask
the question of whether LANA activates the hTERT promoter via a
GC-box-mediated interaction, a probe was designed encompassing a single
GC-box. The sequence for the double-stranded DNA probe was taken from
119 to
98 of the hTERT promoter and was chosen because this GC-box
matches exactly the consensus Sp1 binding sequence. By electrophoretic
mobility shift assay, the probe interacted with BJAB nuclear extracts
to yield at least two GC-box-specific bands (Fig.
7, arrows on left).
These bands were designated as specific shifts because they both
disappeared with GC-box mutant probe (Fig. 7, right panel).
Additionally, these two specific complexes were competed by the
addition of 200× specific competitor; however, these bands were not
competed by nonspecific DNA competitor (Fig. 7, left panel).
Furthermore, one of the specific bands was supershifted with Sp1
monoclonal antibody (Fig. 7, asterisk). In the presence of
in vitro translated LANA, this Sp1-specific signal was
significantly ablated along with the other GC-box-specific bands (Fig.
7, arrows). This ablation was expected because a new LANA
complex would likely be too large to migrate significantly on the gel.
It should be noted that these GC-box-specific signals were not affected
by the addition of nonspecific competitor protein. The small
inset at the left of Fig. 7 shows the LANA-specific
signal from the in vitro translated LANA used in this assay.
Taken as a whole, these data suggest that the endogenous DNA-protein
complex formed at the GC-box and including Sp1 is targeted by LANA.
View larger version (83K):
[in a new window]
Fig. 7.
LANA targets the Sp1-DNA complex. The
probe for the left panel consisted of an Sp1 consensus
sequence (GC-box) and flanking sequence taken from 119 to
98 of the
hTERT promoter; this probe represents one of five Sp1 binding sites
from
130 to +5 of the hTERT promoter. First and
second lanes, probe with or without BJAB nuclear
extract; third and fourth lanes, probe with
nuclear extract and Sp1 mouse monoclonal IgG as indicated;
fifth and sixth lanes, probe with nuclear extract
and either unprogrammed rabbit reticulocyte lysate or in
vitro translated LANA; seventh and eighth
lanes, probe with nuclear extract and 200-fold molar excess either
specific or irrelevant cold probe. The probe for the right
panel differed from the wild-type probe used in the left
panel by a CC to AA mutation in the Sp1 consensus sequence. The
additions to the right panel were as described for the
left panel. The inset at the lower
left shows in vitro translation of pA3M-LANA
(lane 2) in the presence of 35S-labeled Cys and
Met versus negative control (lane 1). The
arrows on the left indicate the positions of the
GC-box-specific shifts. The asterisk indicates the position
of the supershifted complex in the presence of the Sp1 antibody.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
130 to +5 region of the
hTERT promoter in LANA-mediated activation. This region was important
in that it contained five GC-rich boxes, the consensus binding sites
for transcription factor Sp1. Electrophoretic mobility shift assays
were performed to assess the interaction of LANA using a probe
containing a selected GC-box. The data presented here suggest that LANA
alters a specific interaction between Sp1 and the GC-box. However,
neither a LANA-specific shift nor a supershifted complex in the
presence of LANA was observed. Although Sp1, a 558-amino acid protein,
was amenable to gel shift assays resulting in specific shifted
complexes and supershifted complexes, LANA, a 1162-amino acid protein,
would likely be difficult to assay by this method. One explanation for
our data is that LANA interacts with the Sp1-DNA complex, creating a
new complex that is too large to migrate significantly on the gel.
Alternatively, LANA might be recruited to the GC-box displacing the Sp1
complex, creating new complexes and releasing the bound DNA. It should,
however, be noted there was no specific interaction between LANA and
the GC-box in the absence of BJAB nuclear extract. Overall, these data
suggest that the endogenous DNA-protein complex formed at the GC-box is
targeted by LANA, potentially implicating the GC-box in LANA-mediated
activation of the hTERT promoter.
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ACKNOWLEDGEMENTS |
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We thank Elliott Kieff and George Mosialos for the BJAB and early passage Rat-1 cell lines. The hTERT promoter and truncations cloned in the luciferase reporter were kindly provided by J. Carl Barrett. The SUSM-1 cell line was obtained from Masayoshi Namba. 293 and 293T cell lines were obtained from Ian Aster and Jeffrey Sklar.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant NCI c072150-01 (to E. S. R.) and by the Lymphoma and Leukemia Society of America.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by Medical Scientist Training Program Grant T32 GM07863 to the University of Michigan.
Fellow of the Lady Tata Memorial Trust.
Scholar of the Lymphoma and Leukemia Society. To whom
correspondence should be addressed: Univ. of Michigan Medical School, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0620. Tel.:
734-647-7296; Fax: 734-764-3562; E-mail: esrobert@umich.edu.
Published, JBC Papers in Press, April 19, 2001, DOI 10.1074/jbc.M101890200
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ABBREVIATIONS |
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The abbreviations used are: hTERT, human telomerase reverse transcriptase; KS, Kaposi's sarcoma; KSHV, Kaposi's sarcoma-associated herpesvirus; LANA, latency-associated nuclear antigen; HEK, human embryonic kidney; HIV, human immunodeficiency virus; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate.
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REFERENCES |
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