From the Departments of Pathology and
§ Human Genetics, Massey Cancer Center, Medical College of
Virginia at Virginia Commonwealth University, Richmond, Virginia
23298-0662
Received for publication, January 31, 2001, and in revised form, March 21, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The ribonucleoprotein telomerase
holoenzyme is minimally composed of a catalytic subunit, hTERT, and its
associated template RNA component, hTR. We have previously found two
additional components of the telomerase holoenzyme, the chaperones p23
and heat shock protein (hsp) 90, both of which are required for
efficient telomerase assembly in vitro and in
vivo. Both hsp90 and p23 bind specifically to hTERT and influence
its proper assembly with the template RNA, hTR. We report here that the
hsp70 chaperone also associates with hTERT in the absence of hTR and
dissociates when telomerase is folded into its active state, similar to
what occurs with other chaperone targets. Our data also indicate that
hsp90 and p23 remain associated with functional telomerase complexes,
which differs from other hsp90-folded enzymes that require only a
transient hsp90·p23 binding. Our data suggest that components of the
hsp90 chaperone complex, while required for telomerase assembly, remain associated with active enzyme, which may ultimately provide critical insight into the biochemical properties of telomerase assembly.
Vertebrate telomeres are composed of the repeated sequence TTAGGG
and are responsible for maintaining chromosomal stability and integrity
(1). Conventional DNA polymerases are incapable of replicating to the
end of a linear molecule (the end replication problem), resulting in
loss of telomeric DNA during cellular proliferation of normal somatic
cells (2, 3). The specialized reverse transcriptase, telomerase,
compensates for this loss of telomere sequence and is responsible for
maintenance and preservation of telomere ends in germ cells and
immortal and cancer cells (4, 5). The reverse transcriptase subunit of
telomerase, hTERT, contains the catalytic activity of the enzyme,
whereas the associated RNA component, hTR, serves as the template for
synthesis of telomeric sequences (6-8). As direct evidence that
telomere erosion plays a major role in cellular senescence, hTERT was
ectopically expressed in normal human cells, which endogenously express
hTR, resulting in activation of telomerase, stabilization of telomere
lengths, and extension of cellular life span (9, 10).
Expression of the hTERT and hTR components in heterologous systems has
allowed for increased understanding of the biochemical features of the
telomerase enzyme. Reconstitution of human telomerase activity has been
accomplished in a variety of in vitro systems including
yeast, baculovirus, rabbit reticulocyte, wheat germ, and human (6, 7,
11, 12), and each has verified the essential role of hTERT and hTR in
active telomerase. Recently, we have demonstrated that the
hsp901 chaperone complex is
required for assembly of human telomerase both in vitro and
in vivo in a rabbit reticulocyte system and in human cells
(13). We observe that hsp90 and p23 associate with hTERT in the absence
of hTR and that the minimal components necessary for active telomerase
assembly are hTERT, hTR, hsp90, p23, hsp70, p60, and hsp40/ydj.
In addition, we demonstrate that geldanamycin, a potent inhibitor of
hsp90 function, is capable of blocking the assembly of human telomerase
in the rabbit reticulocyte system, as well as in cultured cells.
Recently, additional groups reported on the importance of factor(s) in
the reticulocyte lysate system for telomerase assembly (11, 12, 14),
without specifically identifying the components critical for telomerase assembly.
To date, the hsp90-related chaperone complex is the only set of
proteins that has been shown to functionally associate with human
telomerase and affect its assembly. Here, we report that hsp90 and p23
appear to remain associated with functional telomerase and that hsp70
also associates with hTERT in a transient fashion. Our results
demonstrate that the chaperone/telomerase interaction is critical for
ribonucleoprotein assembly and the formation of active telomerase enzyme.
Plasmids and Gene Expression--
For in vitro
expression, the human hTERT cDNA was inserted into pcDNA3/HisC
(Invitrogen) in frame with three tandem copies of a C-terminal
hemagglutinin epitope (HA). hTERT was synthesized in the rabbit
reticulocyte lysate system (TnT; Promega) as previously described (6)
in the presence of [35S]methionine. pTRC3 was described
previously (6). hTR was produced using pTRC3 (8) with the Megascript T7
in vitro transcription system (Ambion).
Antibodies and Purified Proteins--
Monoclonal anti-HA
antibody (12CA5) was purchased from Roche Molecular Biochemicals.
Monoclonal anti-p23 antibody (JJ3), monoclonal anti-hsp90 antibody
(H9010), and monoclonal anti-hsp70 antibody were described previously
(15, 16). Monoclonal TCP-1 was purchased from Stressgen (13).
Human hsp90 In Vitro Telomerase Assembly--
To assemble active telomerase,
0.2 µl of in vitro-transcribed and -translated
hTERT and 0.5 µg of hTR were mixed together in a 4-µl assembly
assay with or without additional fresh RRL and incubated at 30 °C
for 90 min. For some experiments, hTERT and hTR were coexpressed in the
TnT system, followed by immunoprecipitation (see below) or
reconstitution of activity. Addition of purified chaperones (hsp90 and
p23) were in the amounts of 750 and 500 ng, respectively.
Telomerase Activity Assays--
Telomerase activity in all
samples was determined by TRAP as previously described (6) with minor
modifications. The TRAP-eze telomerase detection kit (Intergen,
Gaithersburg, MD), which includes a 36-bp internal standard to allow
quantitation of activity, was used (17). After telomerase extension for
30 min at room temperature, extended products were amplified by a
two-step PCR (94 °C for 30 s, 60 °C for 30 s) for 27 cycles. These PCR-amplified products were separated by 10%
polyacrylamide gel electrophoresis and exposed to PhosphorImager
screens (Molecular Dynamics, Sunnyvale, CA). Quantitative estimates of
telomerase activity were calculated using ImageQuant software by
determining the ratio of the 36-bp internal standard to the 6-bp
telomerase-specific ladder observed on the gel (17).
Immunoprecipitations--
For immunoprecipitation from in
vitro assembly reactions, antibodies were added to a final
concentration of 0.5 µg/ml and incubated for 1 h on ice. Protein
G-agarose (Roche Molecular Biochemicals) was added, and the mix was
incubated at 4 °C for an additional hour with constant agitation.
Agarose pellets were subsequently pelleted and washed three times with
20 mM HEPES (pH 7.6), 20% glycerol, 100 mM
NaCl, 0.2 mM EGTA, 1 mM MgCl2,
0.1% Nonidet P-40, and 0.1% bovine serum albumin. To detect
proteins, washed pellets were heated to 80 °C for 10 min and
electrophoresed by SDS-PAGE (7.5%); dried gels were exposed to a
PhosphorImager screen (Molecular Dynamics) for 24-48 h. For TRAP
assays following immunoprecipitations, protein G-agarose pellets were
resuspended in a final volume of 8 µl with wash buffer, and 2 µl
were removed for TRAP assay as described. The "double
immunoprecipitation" was done as above except that 4 µl of the
slurry was taken out for traditional TRAP, and the remainder was
subjected to extension of a 32P-labeled primer (from
the TRAP-eze kit) for 60 min at room temperature under typical TRAP
extension conditions. The samples were then reprecipitated using the
residual antibody-protein G-agarose complex, washed extensively as
above, and subjected immediately to the TRAP-eze assay without
telomerase-mediated primer extension.
In Vitro Properties of C-terminal HA-tagged hTERT--
hTERT was
expressed using the RRL system with 2 constructs,
pcDNA3.1-hTERT and pcDNA3.1-hTERT/HA3. For
efficient immunoprecipitation and detection purposes, the latter has a
triple hemagglutinin epitope tag at the C terminus of the expressed
protein, which does not have the ability to elongate telomeres and
extend the life span of normal human fibroblasts (18). To determine
whether there are differences in expression and functionality in our
in vitro reconstitution system, we tested the C-terminal
tagged hTERT against hTERT without a C-terminal tag and found that both
are expressed at consistent levels and have similar amounts of
reconstituted telomerase activity (Fig.
1). In addition, both hTERT molecules appear to associate with the molecular chaperones hsp90 and p23 (data
not shown) as previously described (13). Consistent with our prior
experiments where reconstitution with additional RRL was used as a
source of chaperone proteins, we observe an enhanced activity after
adding RRL using the HA-tagged hTERT (Fig.
2), similar to what was observed for
other hTERT molecules (13). Interestingly, our previous data suggested
that we needed all five chaperone proteins (hsp90, p23, hsp70,
p60, and hsp40/ydj) to establish sufficiency for enhanced
reconstituted activity (13). Fig. 2 shows that addition of only hsp90
and p23 to the minimal reconstitution mix results in an increase in
activity, suggesting that these chaperones may be limiting in the
overall assembly reaction. Thus, because C-terminally tagged hTERT has
nearly identical properties in our in vitro assembly system
to the hTERT without a C-terminal tag, the hTERT/HA3
protein was used for all subsequent experiments.
Association of hsp70 with hTERT--
Other reverse transcriptase
proteins that associate with chaperone proteins do so in a transient
fashion (19, 20); that is, hsp90/p23/hsp70 associate to assist in the
proper folding and conformation of the protein but are not associated
with active reverse transcriptase enzyme. We have already shown that
hsp90 and p23 directly associate with hTERT in our in vitro
system in the absence of hTR (13), and based on previous work, hsp90
and p23 would dissociate from hTERT upon the addition of hTR to our reconstitution system. Our previous data suggest that under optimal conditions, about 50% of the hTERT is bound to hTR in our
reconstitution system (data not shown). Thus, if the chaperone/hTERT
interaction were transient, addition of hTR to the reaction would
result in a reduction in the amount of hTERT precipitated using p23 or
hsp90 antibodies. However, we find just the opposite effect; the data presented in Fig. 3A indicate
that hsp90 and p23 remain associated with hTERT when complexed with
hTR.
In the absence of hormone, steroid hormone receptors associate with
hsp90, p23, and hsp70 (16). We have previously established sufficiency
for telomerase assembly components in that the hsp90 chaperone
machinery can provide the same enhanced telomerase assembly/activity that addition of RRL does in our enhanced reconstitution assay (13).
The five hsp90-related proteins that functionally associate to assemble
human telomerase are hsp90, p23, hsp70, p60, and hsp40/ydj-1. With this
knowledge in hand, our goal was to determine the nature of the hsp70
requirement in telomerase assembly. We found that hsp70 bound
specifically to hTERT in the absence of hTR but that the hsp70 binding
is reduced once hTR is complexed with hTERT, suggesting more of a
transient hsp70 association with telomerase (Fig.
3B).
Stable Association of hsp90 and p23, but Not hsp70, with Functional
Telomerase Enzyme--
The experiments in Fig. 3 suggest that hsp90
and p23 are present in telomerase complexes and that hsp70 is only
transiently associated with hTERT rather than the assembled telomerase
complex. However, this does not address whether hsp90 and p23 interact with active enzyme, only that they are associated with the
hTERT·hTR complex. To more directly address this issue, we
have devised a protocol for specific immunoprecipitation of proteins
associated with functionally active telomerase by precipitating
telomerase-extended products, a method we call double
immunoprecipitation (Fig. 4A). This procedure involves the reconstitution of telomerase using our
in vitro reconstitution system and standard
immunoprecipitation of activity using antibodies directed against
hsp90, p23, and hsp70, as well as control antibodies (the chaperonin
TCP-1) (13). A portion of that precipitation is then assayed for
telomerase activity using the TRAP assay, and the remainder of the
reaction is subjected to telomerase-mediated extension of a telomerase substrate primer under normal TRAP assay conditions. These extended products are then reprecipitated and subjected to the PCR portion of
the TRAP assay without additional extension time (Fig. 4A). In other words, the precipitated telomerase is allowed to extend, and
the telomerase-extended products are reprecipitated using chaperone
antibodies to determine which, if any, are associated with active
enzyme.
As expected, p23 and hsp90 antibodies are capable of precipitating
active telomerase, whereas the hsp70 antibody and the control antibodies, TCP-1 and an antibody specific for rat hsp90, do not pull
down any telomerase activity (Fig. 4B, left
panel). This confirms that although hsp70 associates with hTERT in
the absence of hTR, the association is transient, as hsp70 antibodies
are incapable of precipitating active telomerase. Precipitation of telomerase activity using the single immunoprecipitation approach alone
does not prove that p23 and hsp90 remain associated with active
telomerase, as assembly could occur during the extension step prior to
the PCR amplification of telomerase-extended products. To rule out that
possibility, the double immunoprecipitation shows that hsp90 and p23
appear to remain associated with functional enzyme in that they are
capable of precipitating telomerase-extended products in our in
vitro reconstitution system (Fig. 4B, right panel). Again, neither control nor hsp70 antibodies are able to precipitate active telomerase, a result that was expected based on the
single immunoprecipitation. Interestingly, we consistently observe less
precipitated telomerase activity using the hsp90 antibody than using
the p23 antibody, but the level of activity remains significantly
higher than background controls (Fig. 4B, right
panel).
The hsp90 chaperone complex is currently the only known set of
proteins that functionally interacts with telomerase to induce proper
assembly of the enzyme. In addition to a direct interaction of hsp90
and p23 with telomerase, we show here that hsp70 specifically interacts
with the catalytic subunit, hTERT, in the absence of template RNA, hTR,
and that the hsp70/telomerase interaction appears transient. Other
reverse transcriptases from viral origins associate with hsp70, hsp90,
and p23 in a transient fashion, as well, although there is some
evidence that the viral particle contains hsp90 and p23 (19, 20).
However, what makes the interaction of the molecular chaperones with
the telomerase reverse transcriptase more unique is the apparent stable
association of hsp90 and p23 with functioning telomerase. Our working
model suggests that although hsp90, p23, hsp70, and perhaps other
chaperone proteins are associated with hTERT in its unassembled or
inactive form, the assembled, fully functional telomerase molecule
contains both p23 and hsp90 (Fig. 5).
Although the exact stoichiometry is currently unknown, hsp90 is known
to bind as a dimer to other chaperone targets (21).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and human hsp70 (hsp70A) were purified from baculovirus
preparations, and human p23 was expressed and purified from
Escherichia coli as before (13).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (36K):
[in a new window]
Fig. 1.
In vitro reconstitution of
telomerase activity using hTERT and hTERT/HA3.
A, SDS-PAGE of hTERT and hTERT/HA3 expressed in
the TnT system. The hTERT/HA3 construct with the additional
HA epitopes is slightly larger than the hTERT without a C-terminal tag.
B, telomerase activity was reconstituted using either hTERT
or hTERT/HA3 and subjected to the TRAP assay. Shown are
dilutions of the reconstituted material, with volumes corresponding to
the amount of actual hTERT or hTERT/HA3 TnT mix used in the
assay. C, quantitation of relative telomerase activity for
the assayed volumes of hTERT or hTERT/HA3. Relative
activity is calculated by taking the ratio of the telomerase-specific
ladder to the internal control, and all quantitation was normalized to
0.5 µl of sample (calculated as 1). The data shown represents the
average of three independent experiments.
View larger version (35K):
[in a new window]
Fig. 2.
Enhanced telomerase activity using
recombinant hsp90 and p23 alone. The assembly of telomerase
activity was accomplished with hTERT/HA3 and hTR, yielding
the basal activity level shown in lane 1. No activity was
observed in the absence of hTR (not shown; see Refs. 6 and 13). The
basal assembly reaction was supplemented with either recombinant p23
and hsp90 (lane 2) (13) or rabbit reticulocyte lysate at
50% volume (lane 3). Q denotes quantitation or
-fold activation of telomerase activity.
View larger version (33K):
[in a new window]
Fig. 3.
hsp70 associates with hTERT in a transient
fashion, whereas hsp90 and p23 appear stably associated.
A, hTERT/HA3 was expressed in the TnT system and
immunoprecipitated with HA (positive control;
HA), hsp90 (
hsp90),
or p23 (
p23) antibodies in the absence
(
hTR) or presence (+ hTR) of hTR.
hTERT/HA3 protein was separated by SDS-PAGE and exposed to
the PhosphorImager screen. B, similar to part A,
hTERT/HA3 was immunoprecipitated in the absence or presence
of hTR with
HA, p23 antibody (
p23), or hsp70 antibody
(
hsp70) followed by SDS-PAGE.
View larger version (46K):
[in a new window]
Fig. 4.
Stable association of hsp90 and p23 with
active telomerase enzyme. A, a schematic representation
of the single immunoprecipitation (left side) and the double
immunoprecipitation (right side). The single precipitation
of telomerase activity using chaperone antibodies involves standard
conditions, followed by telomerase extension and the TRAP PCR from
precipitated material. The double IP requires the initial
precipitation, the telomerase-mediated extension while the precipitated
material is on the beads, followed by a second precipitation using the
chaperone antibodies of telomerase-extended products. The products
precipitated in the second round are immediately subjected to the TRAP
PCR without telomerase extension. B, immunoprecipitations of
telomerase activity using chaperone antibodies. The single IP
(left panel; IP only) and the double IP
(right panel; IP, extension, IP) were done using
p23 ( p23), hsp90
(
hsp90), hsp90 (specific for rat hsp90;
hsp90-rat), hsp70
(
hsp70), and TCP-1
(
TCP-1) antibodies. Precipitated products were
PCR-amplified using the TRAP assay and electrophoresed on 10% gels.
The arrow indicates a 36-bp internal control band.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (26K):
[in a new window]
Fig. 5.
Model of chaperone-mediated telomerase
assembly. The model represented indicates the stable
versus transient association of the molecular chaperones
associated with telomerase. In the absence of template RNA
(hTR), the catalytic subunit of telomerase
(hTERT) is complexed to a variety of proteins, including
hsp90, p23, and hsp70, as well as other as yet unidentified proteins.
hTR has an 11-nucleotide template region (boxed) that serves
to associate with the telomere. The model suggests that the hsp90
chaperone complex serves to recruit hTR to hTERT to form an active
enzyme. Our data indicate that hsp90 and p23 remain associated with the
active telomerase, whereas hsp70 is only associated with hTERT in its
inactive form (in the absence of hTR).
The question remains: why would these chaperones be associated with already assembled telomerase? The answer may lie in the reverse transcription process of the telomerase enzyme. Telomerase utilizes its associated RNA component as a template for synthesis of telomeric DNA, and this replication elongates the 3' overhang at the telomere, generally in 6-base increments, for conventional DNA polymerases to replicate further out on the linear chromosome. Thus, telomerase recognizes and elongates the telomere through association with the hTR template region and then translocates to the next available position for hTR binding. It is this translocation step that may require additional "tweaking" of conformation of the assembled complex, which would be provided by the stably associated hsp90 and p23. One interesting caveat is the cellular location of the telomerase assembly process with most of the hsp90, p23, and hsp70 being cytoplasmic and barely 1-2% nuclear (21). This suggests that the overall telomerase assembly process occurs in the cytoplasm but that 1-2% of detectable nuclear hsp90·p23 may be serving to adjust telomerase conformation while associated with the telomere.
Our data directly demonstrate the association of p23 and hsp90 with functional enzyme; therefore, our model simplistically addresses the final step in the assembly of telomerase. Clearly, there are additional steps in the association process that occur prior to the inactive form shown in Fig. 5 and may include an ordered assembly with certain chaperone components. Many other chaperone targets, including steroid hormone receptors, require the ability of hsp70 to provide energy to the hsp90·p23 portion of the reaction (16, 22, 23), a process that occurs prior to the hsp90·p23 association and requires ATP hydrolysis. Our in vitro assembly/reconstitution system will be useful in defining the kinetics and order of chaperone-mediated telomerase assembly, which is critical for understanding telomerase function.
Because telomerase is associated with the vast majority of human malignancies (24), inhibition of telomerase activity may be an attractive molecular target for specific cancer therapy. In fact, telomerase activity has been inhibited by a variety of molecular methods, and the result of that inhibition has been either a delayed senescence phenotype or programmed cell death (apoptosis). Use of dominant-negative hTERT mutants, which contain a mutation in one of the reverse transcriptase motifs rendering the expressed protein devoid of activity, has shown an immediate down-regulation of telomerase activity in tumor-derived cells, followed closely by apoptosis (25, 26). Given that the dominant-negative hTERT mutant is overexpressed in these ectopic systems, one of the mechanisms for telomerase inhibition may be the preferential association of molecular chaperones to the overexpressed mutant rather than the wild-type hTERT, mainly because of the increased number of mutant molecules. This inhibition would prevent the assembly and function of wild-type telomerase, leading to a reduction in activity and ultimately senescence or apoptosis.
Understanding the molecular events leading to telomerase assembly and
function are important in designing effective therapeutic strategies
directed against telomerase and its interacting/modulating proteins.
The hsp90 chaperone complex has been touted as a potential anti-cancer
target, with certain ansamycin compounds (geldanamycin, radicicol, and
herbimycin A) capable of interacting with hsp90 to reduce the
association with ATP and p23, thereby blocking its function (21). One
obvious problem with this approach is the nonspecific toxicity
associated with such therapy, as hsp90 function is required for both
cancer and normal cell function. Recently, there have been additional
geldanamycin analogs that have shown promise in selectively inhibiting
growth of breast cancer cells (27). Using the knowledge of the
chaperone/telomerase interaction may allow for directed therapy against
the assembly of functional telomerase, which may be useful as a
potential adjuvant chemotherapy in the treatment of many types of human cancers.
![]() |
ACKNOWLEDGEMENTS |
---|
We remain grateful to Dr. David O. Toft of the Mayo Clinic (Rochester, MN) for continuous support and for hsp90, p23, and hsp70 antibodies. The hTERT and hTR cDNAs were provided by the Geron Corporation (Menlo Park, CA). We also thank Joe Baur and Dr. Michael White from the University of Texas Southwestern (Dallas, TX) for helpful discussions and participation in this project.
![]() |
FOOTNOTES |
---|
* This work was supported by the V foundation (to S. E. H.), the Mary Kay Ash Foundation (to S. E. H.), and the Howard Hughes Medical Institute (to J. L. J.).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.
¶ To whom correspondence should be addressed: Dept. of Pathology and of Human Genetics, Massey Cancer Center, Medical College of Virginia at Virginia Commonwealth University, 1101 E. Marshall St., Richmond, VA 23298-0662. Tel.: 804-827-0458; Fax: 804-828-5598; E-mail: seholt@ hsc.vcu.edu.
Published, JBC Papers in Press, March 23, 2001, DOI 10.1074/jbc.C100055200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: hsp, heat shock protein; RRL, rabbit reticulocyte lysate; HA, hemagglutinin; TnT, transcription and translation; TRAP, telomeric repeat amplification protocol; IP, immunoprecipitation; bp, base pair; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | de Lange, T., Shiue, L., Meyers, R. M., Cox, D. R., Naylor, S. L., Killery, A. M., and Varmus, H. E. (1990) Mol. Cell. Biol. 10, 518-527[Medline] [Order article via Infotrieve] |
2. | Harley, C. B., Futcher, A. B., and Greider, C. W. (1990) Nature 345, 458-460[CrossRef][Medline] [Order article via Infotrieve] |
3. | Hastie, N. D., Dempster, M., Dunlop, M. G., Thompson, A. M., Green, D. K., and Allshire, R. C. (1990) Nature 346, 866-868[CrossRef][Medline] [Order article via Infotrieve] |
4. | Counter, C. M., Avilion, A. A., LeFeuvre, C. E., Stewart, N. G., Greider, C. W., Harley, C. B., and Bacchetti, S. (1992) EMBO J. 11, 1921-1929[Abstract] |
5. | Kim, N. W., Piatyszek, M. A., Prowse, K. R., Harley, C. B., West, M. D., Ho, P. L., Coviello, G. M., Wright, W. E., Weinrich, S. L., and Shay, J. W. (1994) Science 266, 2011-2015[Medline] [Order article via Infotrieve] |
6. | Weinrich, S. L., Pruzan, R., Ma, L., Ouellette, M., Tesmer, V. M., Holt, S. E., Bodnar, A. G., Lichtsteiner, S., Kim, N. W., Trager, J. B., Taylor, R. D., Carlos, R., Andrews, W. H., Wright, W. E., Shay, J. W., Harley, C. B., and Morin, G. B. (1997) Nat. Genet. 17, 498-502[Medline] [Order article via Infotrieve] |
7. | Beattie, T. L., Zhou, W., Robinson, M., and Harrington, L. (1998) Curr. Biol. 8, 177-180[Medline] [Order article via Infotrieve] |
8. | Feng, J., Funk, W. D., Wang, S.-S., Weinrich, S. L., Avilion, A. A., Chiu, C.-P., Adams, R. R., Chang, E., Allsopp, R. C., Yu, J., Le, S., West, M. D., Harley, C. B., Andrews, W. H., Greider, C. W., and Villeponteau, B. (1995) Science 269, 1236-1241[Medline] [Order article via Infotrieve] |
9. |
Bodnar, A. G.,
Ouellette, M.,
Frolkis, M.,
Holt, S. E.,
Chiu, C.-P.,
Morin, G. B.,
Harley, C. B.,
Shay, J. W.,
Lichtsteiner, S.,
and Wright, W. E.
(1998)
Science
279,
349-352 |
10. | Vaziri, H., and Benchimol, S. (1998) Curr. Biol. 8, 279-282[Medline] [Order article via Infotrieve] |
11. |
Bachand, F.,
and Autexier, C.
(1999)
J. Biol. Chem.
274,
38027-38031 |
12. |
Masutomi, K.,
Kaneko, S.,
Hayashi, N.,
Yamashita, T.,
Shirota, Y.,
Kobayashi, K.,
and Murakami, S.
(2000)
J. Biol. Chem.
275,
22568-22573 |
13. |
Holt, S. E.,
Aisner, D. L.,
Baur, J.,
Tesmer, V. M.,
Dy, M.,
Ouellette, M.,
Toft, D. O.,
Trager, J. B.,
Morin, G. B.,
Wright, W. E.,
Shay, J. W.,
and White, M. A.
(1999)
Genes Dev.
13,
817-827 |
14. |
Licht, J. D.,
and Collins, K.
(1999)
Genes Dev.
13,
1116-1125 |
15. |
Barent, R. L.,
Nair, S. C.,
Carr, D. C.,
Ruan, Y.,
Rimerman, R. A.,
Fulton, J.,
Zhang, Y.,
and Smith, D. F.
(1998)
Mol. Endocrinol.
12,
342-354 |
16. | Johnson, J. L., Beito, T. G., Krco, C. J., and Toft, D. O. (1994) Mol. Cell. Biol. 14, 1956-1963[Abstract] |
17. | Holt, S. E., Norton, J. C., Wright, W. E., and Shay, J. W. (1996) Methods Cell Sci. 18, 237-248 |
18. | Ouellette, M. M., Aisner, D. L., Savre-Train, I., Wright, W. E., and Shay, J. W. (1999) Biochem. Biophys. Res. Commun. 254, 795-803[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Hu, J.,
and Seeger, C.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
1060-1064 |
20. |
Hu, J.,
Toft, D. O.,
and Seeger, C.
(1997)
EMBO J.
16,
59-68 |
21. | Neckers, L., Mimnaugh, E., and Schulte, T. W. (1999) Drug Res. Upd. 2, 165-172[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Hutchison, K. A.,
Dittmar, K. D.,
Czar, M. J.,
and Pratt, W. B.
(1994)
J. Biol. Chem.
269,
5043-5049 |
23. | Toft, D. O. (1998) Trends Endocrinol. Metab. 9, 238-243[CrossRef] |
24. | Shay, J. W., and Bacchetti, S. (1997) Eur. J. Cancer 33, 787-791[CrossRef][Medline] [Order article via Infotrieve] |
25. | Hahn, W. C., Stewart, S. A., Brooks, M. W., York, S. G., Eaton, E., Kurachi, A., Beijersbergen, R. L., Knoll, J. H., Meyerson, M., and Weinberg, R. A. (1999) Nat. Med. 5, 1164-1170[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Herbert, B.,
Pitts, A. E.,
Baker, S. I.,
Hamilton, S. E.,
Wright, W. E.,
Shay, J. W.,
and Corey, D. R.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
14276-14281 |
27. |
Zheng, F. F.,
Kuduk, S. D.,
Chiosis, G.,
Munster, P. N.,
Sepp-Lorenzino, L.,
Danishefsky, S. J.,
and Rosen, N.
(2000)
Cancer Res.
60,
2090-2094 |