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Advances in Brief |
Departments of Pathology [G. H. S.] and Surgery [T. A. S.] and The Oncology Center [G. H. S., B. R., S. E. K.], The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205-2196
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Currently, the common methods used to identify the mediator and regulators of a pathway involve physical interactions (two-hybrid analysis and protein affinity chromatography), experimental genetic means (mutagenesis of bacteria or yeast), and observations of natural genetic variance (human tumors and inherited disease susceptibility). Cloning by physical interactions is rather inefficient and laborious and can be biased by the initial choice of the bait. Experimental genetic methods are efficient and unbiased but difficult to apply to mammalian cells and human disease. Research based on natural genetic variance is highly relevant to the understanding of human disease, but the sample size of such an approach is often limited. The screening of random libraries of chemical compounds can encompass all of the benefits of current methodologies; they can be unbiased and high-throughput and can be used to probe and dissect complex biological systems in mammalian cells (1 , 2) . Screening of compound libraries can also directly produce candidates for therapeutic and experimental applications.
The "true" activation of a signaling or regulatory pathway can be difficult to measure. The detection of a downstream transcriptional event using a specific reporter construct in cells is a common method. This type of manipulation of a pathway requires a basic understanding of the participating members of the pathway, but the understanding is often incomplete and imprecise. To test a promoter or an enhancer under adequate sensitivity, the selection of reporter genes often is limited. The interpretation of results using downstream reporter constructs can be misled by unanticipated positive or negative influences. That is, it is often difficult to distinguish release of inhibition from true activation, and vice versa. Furthermore, the system depends on the recruitment of the general transcriptional apparatus, the interaction of which with the system under study is seldom known in advance.
We chose to address these issues by the implementation of a high-throughput compound screening, using a stably integrated reporter construct to identify reliable and important regulators of a tumor-suppressive pathway. The TGFß3 pathway is well studied biologically and comprises a number of human tumor-suppressor genes, including SMAD4 (MADH4, DPC4; Refs. 3, 4, 5 ). Our reporter construct contains Smad-binding elements (p6SBE-luc) and allows us to measure processes that result in the nuclear localization of Smad4 (6) . The discovery of agents that would interact with or bypass deficits in the TGFß pathway by augmenting the action of downstream Smad4 would likely be useful to the biological understanding of this tumor-suppressive pathway.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Cell Lines.
PANC-1 and MDA-MB-468 cell lines were purchased from American Type
Culture Collection (Manassas, VA). Stable transfectants were generated
by cotransfection of pcDNA3.1 (Invitrogen, Carlsbad, CA) and p6SBE-luc
into PANC-1 cells with lipofectamine (Life Technologies, Inc.).
Transfected cells were diluted and selected in multiple 96-well plates
in the presence of 0.5 mg/ml of G418 (Life Technologies, Inc.). Single
clones were expanded and tested for basal luciferase expression and
TGFß inducibility. One clone was chosen on the basis of high (6- to
8-fold) induction of luciferase by 0.5 ng/ml TGFß (R&D Systems,
Minneapolis, MN).
Compound Screening.
Each compound of the library (DIVERSet, ChemBridge, San Diego, CA) was
dissolved and diluted in DMSO at 1 mg/ml. Cells were plated in 96-well
cluster plates (Corning, Cambridge, MA) and incubated with each
compound, after further dilution in culture medium to the final
concentration of 2 µg/ml, for 16–18 h. Luciferase activity was
measured on the addition of Steady-Glo substrate (Promega). Up to 16
96-well plates could be assembled in a Wallac Trilux photodetector
(Wallac, Gaithersburg, MD) for measurement. All of the readouts from
each experiment were compared with the control wells, and a number
reflecting the relative increase in luciferase activity was calculated
for each chemical by using Excel (Microsoft, Redmond, WA) spreadsheets.
Immunoblotting Assay of Histone Acetylation.
PANC-1 cells were treated with 2 µg/ml of scriptaid (ChemBridge) or
0.1 to 0.32 µg/ml of TSA (Sigma, St. Louis, MO) for 18 h in
culture medium. Treated and untreated cells were harvested with
trypsin-EDTA (Life Technologies, Inc.), washed with PBS (Life
Technologies, Inc.), and resuspended in a protein sample buffer.
Protein concentration was determined by BCA protein assay
reagents (Pierce, Rockford, IL). Fifty µg of proteins from each
sample was loaded on a 12% denaturing polyacrylamide gel. Proteins
were subsequently transferred to a nylon membrane (Imobilon P,
Millipore, Burlington, MA) using Milliblot-Graphite Electroblotter I
(Millipore). The nylon membrane was incubated with rabbit antihuman
acetyl-lysine antibody (Upstate Biotechnology, Waltham, MA) at 1:1000
dilution, followed by goat antirabbit antibody coupled to horseradish
peroxidase (Pierce) at 1:2000 dilution, developed with SuperSignal
substrates (Pierce), and detected by film (BioMax, Kodak, Rochester,
NY).
Survival Curve.
Equal numbers of cells were plated in six-well plates in the absence or
presence of scriptaid or TSA at different concentrations. After 18
h of incubation, cell numbers were determined by trypan blue exclusion.
Percent survival of the treated cells was calculated in comparison to
the untreated sample, which was considered to represent 100%.
Transfection Assay.
Each transient transfection experiment was done in duplicate in
six-well plates. Lipofectamin (Life Technologies, Inc.) was used as
directed by the manufacturer. The DNA-lipofectamin mixture was removed
from cells after 4–5 h of transfection, and culture media with or
without compounds or TGFß was then added to the cells. Sixteen to
18 h from the start of the transfection, cell lysates were
prepared with Reporter Lysis Buffer (Promega) for luciferase and
ß-gal assays. Luciferase was measured using The Luciferase
Assay System (Promega) and ß-gal assay was performed as described
previously (6)
. Studies of the SV40 promoter included all
of the experiments performed with p6SBE-luc, p6MBE-luc, and
pGL3-control (Promega) plasmids. Studies of the CMV promoter were done
using pCMVß (Clonetech, Palo Alto, CA), and those of human ubiquitin
c promoter were done using pUB6/V5-lacZ (Invitrogen).
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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). Additional studies on the latter compound (Identification No.
217444;
6-(1,3-dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-hexanoic
acid hydroxyamide, we termed scriptaid) are reported here (Fig. 2
). A related compound (Identification No. 158497;
4-(1,3-dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxy-butyra-mide,
we termed nullscript), which did not induce the p6SBE-luc reporter
construct in the initial screen, was identified from the library using
ChemFinder (Cambridge Soft, Cambridge, MA) by its structural similarity
to scriptaid (Fig. 2
). The results were validated by repeated
determinations in the screening assay and subsequently by a
dose-response curve performed on PANC-1 cells that contained stably
integrated p6SBE-luc (Fig. 3
).
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). The direct interaction of TSA
and a HDAC has been examined by crystallography (7)
. The
hydroxamic acid group of TSA coordinates the zinc atom in the polar
pocket of HDAC in the crystal structure of the
HDAC-Zn2+-TSA complex. The hydroxamic acid group
on TSA is attached to a five-carbon (excluding the carbon elements of
the hydroxamic acid group or the keto group) aliphatic chain that spans
a narrow tube-like pit formed by the surface of HDAC. The double bonds
and the methyl-group in the aliphatic chain of TSA (Fig. 2
) are not
necessary for its inhibitory function (7)
. The bulky
end-group on the opposite end of the aliphatic chain is positioned
outside the entrance to the pit. Similar to TSA, scriptaid has a
five-carbon linker between a bulky end-group and the hydroxamic acid
moiety. Nullscript, which is almost identical to scriptaid except for a
three-carbon (rather than five-carbon) linker (Fig. 2
), was inactive in
transcriptional facilitation at corresponding concentrations (Fig. 3
),
which confirmed a minimal requirement for the length of the linker
chain expected for this class of HDAC inhibitors.
The use of scriptaid resulted in a >100-fold increase in histone
acetylation (Fig. 4
) in cultured cells, which confirmed scriptaid as a HDAC inhibitor.
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). Scriptaid worked
optimally at 2–2.5 µg/ml (6–8 µM), and TSA activity
peaked at 0.32 µg/ml (1 µM). At its optimal
concentration for transcriptional facilitation, scriptaid was not
lethal to one cell line and had limited effects (80% survival) on
another (Fig. 5A
). TSA was cytotoxic for two cell lines at its optimal
concentration range (Fig. 5B
). TSA at its minimal toxic
concentration (0.1 µg/ml; 85–90% survival) was less efficient at
inhibiting endogenous histone deacetylation (Fig. 4
).
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), irrespective of the presence or absence of scriptaid.
Scriptaid could thus facilitate transcription independent of a positive
inducer of transcription, producing multiplicative rises in reporter
activity. Similar effects were seen with either scriptaid or TSA when
the reporter construct was stably integrated into the host cell genome
(Fig. 6, B and C
). Scriptaid and TSA can
proportionally enhance the induction of an integrated p6SBE-luc
construct without interfering with TGFß-stimulated transcriptional
responses.
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), pCMVß (Fig. 7B
), and pUB6/V5-LacZ (Fig. 7C
), driven by viral (SV40 and CMV) or human (ubiquitin c,
UB6) promoters. This general facilitation of transcription by scriptaid
did not depend on the specificity of the enhancer (SBE
versus MBE), the type of promoter (viral versus
cellular), the product of the reporter gene (luciferase
versus ß-gal), nor on the integration status of the
reporter construct (stable versus transient). The ability of
scriptaid to facilitate transcriptional activation was consistently
robust and concentration-dependent in both stable and transient
reporter assays (Fig. 3
and 7
, and data not shown). In contrast, the
performance of TSA was less predictable at its optimal concentration
(Fig. 7, A and B
; see MDA-MB-468).
Lowering the concentration of TSA (0.1 µg/ml) could mitigate this
lack of consistency, but in doing so, the efficiency of TSA as an HDAC
inhibitor or a general transcription facilitator was significantly
compromised (Fig. 4
and 7
).
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Scriptaid is a novel HDAC inhibitor that belongs to an existing class
of hydroxamic acid-containing HDAC inhibitors. Scriptaid possesses a
general property of transcriptional facilitation that applies to stably
integrated or transiently transfected exogenous constructs, to
promoters derived from viruses or an endogenous gene, to multiple
reporter genes, and to different cell lines. Scriptaid does not
interfere with the ability of a reporter construct to measure the
positive (purely inductive) activation of a transcription factor in
response to a known signal transduction stimulus. In relation to other
members of its class, the optimal concentration of scriptaid (6–8
µM) is similar to those reported for suberoylanilide
hydroxamic acid (2 µM) and m-carboxycinnamic
bis-hydroxamide (4 µM; Ref. 11
), higher than
TSA (1 µM, as measured here), and much lower than those
reported for hexamethylenebisacetamide (5000 µM) and
diethyl bis-(penta-methylene-N,N-dimethylcarboxamide)
malonate (400 µM; Ref. 11
). Our data
suggested some advantages of scriptaid over TSA in the range of
promoters subject to predictable effects (Fig. 4
and 7
) and in cellular
toxicity (Fig. 4
and 5
), although some degree of cellular toxicity may
be a general feature of this class of compounds when used at
transcriptionally effective concentrations (11)
.
The ability of scriptaid to indiscriminately facilitate transcriptional activation and its facilitation of detection of a positive transcriptional signal suggest the usage of scriptaid as a useful reagent for transactivation assays in reporter systems, perhaps allowing the use of difficult-to-transfect cells, the use of the available but less sensitive reporter genes, such as green fluorescent protein, or the minimization of culture volumes to aid high-throughput compound or biological screening and for adaptation to robotic handling. A reduction in the signal transduction strength needed to detect the operation of a reporter suggests a utility in the measurement of signal transduction events at a lower and thus more physiological range. For example, the use of scriptaid would be expected to reduce the requirement for protein overexpression or for high (pharmacological) levels of ligand often used to facilitate the evaluation of a signaling pathway. Application to other protein expression methods is also possible.
Currently underappreciated is the strength of the background of transcriptional repression that acts on general-utility promoters. Use of a relative nontoxic HDAC inhibitor such as scriptaid, thus, could conceivably simplify the interpretation of transcriptional reporter assays. We observed that at least 90% of the potential magnitude of the inducible transcriptional activation of our reporter system was originally repressed. It is known that the expression of some genes is regulated by the degree of histone acetylation (12) . Thus, a 2-fold induction seen in an experimental situation may not always represent a 100% increase in strength of transactivation per se but could be mimicked, for example, by a 10% decrease in repression. These two possibilities could presumably be distinguished by the use of scriptaid and other HDAC inhibitors. In some reporter systems, negative effects on transcription (repression) may completely overshadow the positive effects. The use of HDAC inhibition to chemically dissect a pathway should unmask some important measures of pathway activation that could be overlooked in an undissected system. Indeed, it has been previously observed that the presence of TSA or butyrate indeed uncovered the inducibility of certain reporters that initially had appeared inactive (13 , 14) .
TSA and butyrate are the most well studied of the HDAC inhibitors for their effects on reporters or integrated genes. Yet, the potential of such general applications of HDAC inhibitors are somewhat controversial, perhaps because the properties of TSA and butyrate in the published reports had been confusing. Various limitations of TSA and butyrate in the applicability to transcriptional assays have been noted in endogenous genes and on the introduction of exogenous sequences. Butyrate and phenylbutyrate have many functions other than inhibiting HDACs; they have been reported to affect the posttranscriptional modification of other genes (15) and the depletion of glutamine (16) . There are variable observations that conclude that TSA and other inhibitors do not consistently activate all of the promoters, and such failures of transcriptional facilitation have included the common general-utility promoters CMV and SV40 (17, 18, 19) . Some of the reported transcriptional actions required a specific small recognition element (20, 21, 22) , or the activity of a particular coactivator (23) . Furthermore, TSA is not always found to facilitate the detection of positive signal transduction events without interfering with the magnitude of relative transactivation activity (13 , 22) . It was, therefore, of interest that a more general utility could here be indicated, at least for some HDAC inhibitors within a defined system.
In summary, the identification of scriptaid confirmed the feasibility of compound screening in mammalian cells using this reporter system, in the absence of a formal compound-design effort. Scriptaid is shown to be a novel HDAC inhibitor with robust activity and relatively low toxicity, which suggests a wider utility in transactivation assays and in studies of histone acetylation.
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Acknowledgments |
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FOOTNOTES |
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1 Supported by the NIH SPORE (Specialized Program
of Research Excellence) in Gastrointestinal Cancer Grant CA 62924 and
NIH Grant CA 68228. 
2 To whom requests for reprints should be
addressed, at the Department of Oncology, 451 Cancer Research Building,
1650 Orleans Street, The Johns Hopkins University School of Medicine,
Baltimore, MD 21231. Phone: (410) 614-3314; Fax: (410) 614-9705;
E-mail: sk{at}jhmi.edu
3 The abbreviations used are: TGF, transforming
growth factor; HDAC, histone deacetylase; TSA, trichostatin A; SBE,
Smad-binding element; MBE, mutated SBE; ß-gal, ß-galactosidase;
CMV, cytomegalovirus; CBP, CREB-binding protein.
Received 1/ 7/00. Accepted 4/28/00.
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REFERENCES |
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)-induced biosynthesis of MHC class III gene products (complement C4 and factor B) in human fetal intestinal epithelial cells. Clin. Exp. Immunol., 118: 16-22, 1999.[Medline]
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