MOLECULAR AND CELLULAR BIOLOGY, Jan. 2003, p. 655–664
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 23, No. 2
Growth Suppression of Pre-T Acute Lymphoblastic Leukemia Cells by
Inhibition of Notch Signaling
Andrew P. Weng,1,2Yunsun Nam,1,2Michael S. Wolfe,3Warren S. Pear,4James D. Griffin,3,5
Stephen C. Blacklow,1,2and Jon C. Aster1,2*
Departments of Pathology1and Medicine,3Brigham and Women’s Hospital, Department of Pathology,2
Harvard Medical School, and Department of Adult Oncology, Dana-Farber Cancer Institute,5Boston,
Massachusetts 02115, and Department of Pathology and Laboratory Medicine, Institute for
Medicine and Engineering, The Abramson Family Cancer Research Institute, University
of Pennsylvania Medical School, Philadelphia, Pennsylvania 191044
Received 26 July 2002/Returned for modification 20 September 2002/Accepted 21 October 2002
Constitutive NOTCH signaling in lymphoid progenitors promotes the development of immature T-cell
lymphoblastic neoplasms (T-ALLs). Although it is clear that Notch signaling can initiate leukemogenesis, it
has not previously been established whether continued NOTCH signaling is required to maintain T-ALL
growth. We demonstrate here that the blockade of Notch signaling at two independent steps suppresses the
growth and survival of NOTCH1-transformed T-ALL cells. First, inhibitors of presenilin specifically induce
growth suppression and apoptosis of a murine T-ALL cell line that requires presenilin-dependent proteolysis
of the Notch receptor in order for its intracellular domain to translocate to the nucleus. Second, a 62-amino-
acid peptide derived from a NOTCH coactivator, Mastermind-like-1 (MAML1), forms a transcriptionally inert
nuclear complex with NOTCH1 and CSL and specifically inhibits the growth of both murine and human
NOTCH1-transformed T-ALLs. These studies show that continued growth and survival of NOTCH1-trans-
formed lymphoid cell lines require nuclear access and transcriptional coactivator recruitment by NOTCH1 and
identify at least two steps in the Notch signaling pathway as potential targets for chemotherapeutic
Notch signaling plays an important role in diverse cellular
and developmental processes, including differentiation, prolif-
eration, survival, and apoptosis (reviewed in reference 1). For
example, the mammalian NOTCH1 gene has an essential role
in the development of T cells from common lymphoid progen-
itors, as NOTCH1 insufficiency leads to intrathymic B-cell de-
velopment at the expense of T-cell development (43). Con-
versely, inappropriate increases in NOTCH1 signaling cause
ectopic T-cell differentiation within the bone marrow at the
expense of B-cell differentiation (42). Enforced NOTCH1 sig-
naling eventually leads to the development of lethal CD4/
CD8?/?T-cell lymphoblastic neoplasms (T-ALLs) (40), indi-
cating NOTCH functions as an oncoprotein in certain contexts.
Normal NOTCH1 is a heterodimeric type I transmembrane
receptor composed of two polypeptide chains, an extracellular
subunit (NEC) and a transmembrane subunit (NTM), which
are produced by cleavage (S1 in Fig. 1a) of a single precursor
polypeptide by a furin-like convertase (35). The NEC subunit
includes 36 iterated epidermal growth factor (EGF)-like re-
peats that bind ligands of the Delta and Serrate families (45).
Although it is very difficult to detect Notch in the nucleus of
normal cells, numerous genetic and biochemical studies have
converged on a model for signaling in which ligand binding
renders the receptor sensitive to at least two successive pro-
teolytic cleavages (reviewed in reference 38). The first cleavage
occurs just external to the transmembrane domain (S2 in Fig.
1a) and is mediated by metalloproteases of the ADAM family
(7, 35). The second cleavage, which occurs within the inner
portion of the lipid bilayer (S3 in Fig. 1a), releases the intra-
cellular domain of NTM (ICN) from its membrane tether. This
cleavage requires presenilin 1 or 2 (13, 55), members of a
family of novel polytopic transmembrane proteins that likely
function as aspartyl proteases (57, 59). Free ICN then trans-
locates to the nucleus, where it interacts with the DNA binding
transcription factor CSL [named for its murine, Drosophila,
and Caenorhabditis elegans homologs CBF1, Su(H), and Lag-1,
respectively] and with conserved transcriptional coactivators of
the Mastermind family to form a ternary complex that stimu-
lates the transcription of downstream target genes (15, 41, 60).
Although the RAM domain of ICN has been identified as
mediating high-affinity interaction with CSL, the ankyrin re-
peat (ANK) domain also binds weakly (3, 31, 54). The ANK
binding site for CSL may be critically important in vivo, as
RAM-less forms of ICN1 retain the capability to stimulate
transcription from CSL reporters, whereas ANK deletions ren-
der ICN1 nonfunctional (3, 4). The ANK domain also serves as
the binding site for Mastermind-like coactivators (MAMLs)
(15, 41, 60), which interact with ANK through an N-terminal
basic domain (Fig. 1b). Structure and leukemogenesis analyses
have shown that both ANK and a C-terminal transcriptional
activation domain (TAD) are required for induction of T-ALL
in a murine model (4).
Mammalian NOTCH1 was initially identified through anal-
ysis of a recurrent (7;9)(q34;q34.3) chromosomal translocation
found in sporadic human T-ALL (16). The t(7;9) fuses the 3?
* Corresponding author. Mailing address: Brigham and Women’s
Hospital, Department of Pathology, 20 Shattuck St./Thorn 503, Bos-
ton, MA 02115. Phone: (617) 732-7980. Fax: (617) 732-7449. E-mail:
end of NOTCH1 to the T-cell receptor ? promoter/enhancer
and results in the expression of a series of aberrant mRNAs
encoding nuclear forms of NOTCH1 that resemble ICN1, sug-
gesting that T-cell transformation stems from an increase in
signals mediated by dysregulated expression of nuclear
NOTCH1. While transgenes encoding predominantly nuclear
ICN1 induce T-ALL when expressed in murine bone marrow
cells (3, 4, 40), the necessity of nuclear localization for T-cell
transformation has not been formally proven, and certain ob-
servations raise the possibility that alternative signaling mech-
anisms could be relevant. Most directly, an N-terminally de-
leted form of membrane-tethered NOTCH1 (termed ?E) (Fig.
1a) is a potent inducer of T-ALL in the mouse, yet it shows no
detectable evidence of processing to a nuclear derivative as
judged by immunostaining of cells or cell extracts (3, 40). In
addition, some genetic and biochemical data support the exis-
tence of CSL-independent NOTCH signaling pathways that
might proceed through mechanisms not requiring nuclear ac-
cess (8, 27, 37, 39, 44, 51, 56).
Another area of uncertainty is whether persistent NOTCH1
signaling is necessary for tumor growth after transformation
has occurred. Transgenic expression of active Notch isoforms
has been shown both to drive development of immature T cells
from lymphoid precursors and to prevent their further matu-
ration (26, 42), suggesting that NOTCH1 signaling contributes
to transformation by influencing differentiation. It is not
known whether other potential effects of NOTCH signaling,
such as inhibition of apoptosis (12, 29) or enhancement of
proliferation (6, 21), contribute to Notch-mediated transfor-
mation. Determination of whether continued NOTCH1 signal-
ing is required for cell proliferation once tumors have become
established is an issue of central importance when considering
NOTCH1 as a potential therapeutic target in malignancies
such as T-ALL.
By using cell lines derived from NOTCH1-associated T-
ALLs, we show here that presenilin inhibitors suppress the
growth of ?E-expressing cells but not ICN1-expressing cells,
arguing that NOTCH1 nuclear access is required to maintain
tumor cell growth and survival. We then demonstrate that
dominant-negative peptides derived from Mastermind-like-1
(MAML1), which prevent recruitment of coactivators to the
CSL/ICN1 complex, specifically antagonize the growth of mu-
rine ?E- and ICN1-expressing cell lines as well as cells from a
human T-ALL bearing the t(7;9). Together these findings sup-
port a model in which recruitment of transcriptional coactiva-
tors to ICN1 complexes is necessary for the proliferation and
survival of Notch1 leukemia cells.
MATERIALS AND METHODS
Cell lines. The NOTCH1-associated T-ALL lines used have been described
previously (16, 40). All other lines were obtained from the American Type
Culture Collection (Manassas, Va.), including the murine T-lymphoblastic cell
line BW5147 (ATCC designation BW5147.G.1.4). T-ALL cell lines and BJAB
cells were maintained in RPMI (Gibco, Carlsbad, Calif.) supplemented with a
solution containing 10% fetal bovine serum (BioWhittaker, Wakersville, Md.), 1
mM sodium pyruvate (Mediatech, Herndon, Va.), 2 mM L-glutamine (Gibco),
100 U of penicillin G/ml, and 100 mg of streptomycin/ml. U2OS and 293T cells
were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) with
the same supplements. Cells were grown at 37°C under 5% CO2.
Expression plasmids. Constructs for expression of MAML1 and ICN1
polypeptides were created by PCR amplification with human NOTCH1 (16) and
MAML1 (60) cDNAs as templates. Eukaryotic MAML1 expression constructs
were engineered in the plasmid vector pcDNA3 (Invitrogen, Carlsbad, Calif.) to
produce peptides with a C-terminal three-hemagglutinin tag. To permit stable
expression in lymphoid cells, cDNAs encoding MAML1 peptides fused N ter-
minally to green fluorescent protein (GFP) (Clontech, Palo Alto, Calif.) were
cloned into the backbone of the retroviral shuttle vector MSCV-IRES-GFP (42),
replacing the resident internal ribosome entry sequence (IRES) and GFP se-
quences. Bacterial MAML1 expression constructs were engineered in the plas-
mid vector pRSET-A (Invitrogen) to produce peptides with an N-terminal six-
His tag followed by a cleavage site for tobacco etch virus (TEV) protease.
Constructs were designed such that only a single additional glycine residue
remained at the N terminus of encoded MAML1 peptides following cleavage
with TEV protease. Expression constructs for NOTCH1 in pcDNA3, MSCV-
IRES-GFP (4), pET41 (Novagen, Madison, Wis.), myc-tagged CSL in pcDNA3
(3), and MAML1 in CMV2-FLAG (Sigma, St. Louis, Mo.) (60) have all been
Presenilin inhibitors. DFP-AA (also called compound E in the literature) is a
benzodiazepine-type compound and was synthesized as described previously
(50). DAPT is N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl
ester, WPE-III-86 is the unfluorinated counterpart of DAPT, WPE-III-109 is a
truncated version of DAPT lacking the phenylglycine residue, WPE-III-18 is the
methyl ester variant of DAPT, and WPE-III-141 is the Ala-Leu counterpart to
WPE-III-18. All these dipeptide analogues were synthesized as described previ-
ously (14). The remaining compounds are all (hydroxyethyl)urea transition-state
analogues exemplified by WPE-III-31C (17), with the structure Boc-Phe?Phe-
FIG. 1. (a) Schematic representation of various forms of NOTCH
referred to in this study. The normal mature heterodimeric receptor is
produced by cleavage at S1. Cleavages at S2 and S3 are normally
regulated by ligand binding to NEC but occur constitutively in the case
of the ?E form. Cleavage of ?E at S2 by a metalloprotease yields the
?E* form. Cleavage of ?E* at S3 by presenilins releases ICN from the
membrane. ICN1 constructs utilized in T6E/DFP-AA rescue experi-
ments are indicated (ICN, ?RAM?P, and ?TAD?P). P, PEST do-
main. (b) Schematic representation of functional domains of MAML1.
Amino acid residue positions are indicated; 1016 represents full-length
MAML1. ICN, Notch-binding domain; p300, p300 recruitment domain
according to Fryer et al. (20); CoA*, recruitment domain for uniden-
tified transcriptional coactivator(s).
656WENG ET AL.MOL. CELL. BIOL.
Leu-Val-OMe, where ? is the pseudopeptide bond containing the hydroxyethyl
group. MW-III-36A is Boc-Phe?Ala-Leu-Phe-Ome, MW-III-36B is Boc-
Phe?Ile-Leu-Phe-Ome, MW-III-36C is Boc-Phe?Phe-Leu-Phe-Ome, MW-III-
38A is Boc-Phe?Phe-Ala-Phe-Ome, and MW-III-38B is Boc-Phe?Phe-Val-
Phe-OMe. These analogues were all synthesized according to methods described
Reporter gene assays. For the presenilin inhibitor assays, empty pcDNA3 or
pcDNA3-?E (10 ng/well) was transiently transfected in triplicate into human
U2OS cells in 24-well dishes (Falcon 3047) by using Lipofectamine Plus (Invitro-
gen) along with the firefly luciferase reporter CBF1-luc (24) and an internal
Renilla luciferase control plasmid, phRL-TK (Promega, Madison, Wis.). Prese-
nilin inhibitor compounds (1 ?M) were added to the cultures immediately
posttransfection and were added again in fresh media 1 day posttransfection. To
test dominant-negative MAML1 peptides, U2OS cells in 24-well dishes were
transfected with empty pcDNA3 or pcDNA3-ICN1 (10 ng/well) plus various
pcDNA3 plasmids (50 ng/well) encoding MAML1 or MAML1 peptides with
three C-terminal hemagglutinin tags. All dual luciferase assays were performed
and analyzed by using cell extracts prepared 40 to 44 h posttransfection, as
described previously (4).
Retroviral gene transduction. Production of pseudotyped MSCV-GFP viruses
and retroviral spin infections and flow cytometric detection of GFP-expressing
cells were all done as described previously (10, 42). GFP expression appeared by
36 h posttransduction and generally peaked ?72 h posttransduction.
Cell growth assays. Cultured cells were treated with a presenilin inhibitor
compound (DFPAA, WPE III-18, MW III-36A, or WPE III-109) at concentra-
tions of 100 nM to 5 ?M. Viable cell counts were performed daily, either
manually by trypan blue exclusion or with an automated clinical hematology
analyzer (Advia 120 Hematology System; Bayer Diagnostics, Tarrytown, N.Y.).
Viable cells distributed primarily in the lymphocyte and large unstained cell gates
and nonviable cells distributed primarily in the platelet and cell debris gates.
Two- to 3-ml cultures were initially seeded at 7.5 ? 105viable cells/ml in 12- or
24-well dishes and were reseeded each day at the same density in media with
fresh inhibitor. Mock-treated controls were exposed to equivalent concentrations
of carrier (up to 0.05% dimethyl sulfoxide). Extrapolated cell counts were cal-
culated each day by using the formula [(current day’s cell concentration)/(7.5 ?
105cells/ml)] ? (previous day’s extrapolated cell count).
Cell cycle and apoptosis analysis. Cells were stained with either propidium
iodide or DRAQ5 for DNA content measurement. For propidium iodide stain-
ing, cells were washed once in cold phosphate-buffered saline (PBS), fixed in
ice-cold 70% ethanol for at least 24 h at ?20°C, and stained with 40 ?g of
propidium iodide (Sigma)/ml with (100 ?g/ml) DNase-free RNase A in PBS for
30 min at 37°C at a concentration of 5 ? 105cells/ml. For DRAQ5 staining,
DRAQ5 dye (Biostatus, Leicestershire, United Kingdom) was added to live cells
at a final concentration of 10 ?M in complete culture medium and was incubated
at 37°C for 15 min. Immediately after staining, DNA content was measured by
using either a Coulter Epics XL-MCL single-laser 4-color flow cytometer or a
Coulter Cytomics FC 500 dual-laser 5-color flow cytometer (Beckman Coulter,
Miami, Fla.). Intact, single cells were gated by using propidium iodide and
propidium iodide peak fluorescence. For DRAQ5 staining studies, DNA content
was measured in separately gated GFP-deficient and GFP?cell populations. Cell
cycle fractions were determined by using MultiCycle software (Phoenix Flow
Systems, San Diego, Calif.). Apoptotic cells were identified by staining of PBS-
washed, unfixed cells with Annexin-V-phycoerythrin (PE)/7-aminoactinomycin
D (7-AAD) according to the manufacturer’s recommended conditions (BD
Pharmingen, San Diego, Calif.), followed by flow cytometric analysis. Apoptotic
cells were measured in the PE?/7-AAD?gate. Dead cells were excluded as
Immunoprecipitation and Western blotting. Immunoprecipitates were pre-
pared from whole-cell extracts of 5 ? 106cells with a rabbit polyclonal antibody
against NOTCH1, designated ?-TC, as described previously (3). Immunopre-
cipitated proteins on protein A beads (Pharmacia, Piscataway, N.J.) were treated
with lambda protein phosphatase (New England Biolabs, Beverly, Mass.) as
recommended by the manufacturer prior to electrophoresis in discontinuous
sodium dodecyl sulfate polyacrylamide gels. Western blots were stained with
?-TC by using a chemiluminescent method (SuperSignal West Pico; Pierce,
Rockford, Ill.) as described previously (3).
Northern analysis. Total RNA was prepared from PBS-washed tissue culture
cells by using Trizol reagent (Invitrogen) according to the manufacturer’s rec-
ommendations. Two micrograms of total RNA from each sample was electro-
phoresed through 1.2% agarose–2.2 M formaldehyde gels in 1? morpholinepro-
panesulfonic acid buffer, transferred to Nytran SPC membranes (Schleicher &
Schuell, Keene, N.H.) in 10? SSPE (1? SSPE is 0.18 M NaCl, 10 mM NaH2PO4,
and 1 mM EDTA [pH 7.7]), and UV cross-linked by using a Stratalinker (Strat-
agene, La Jolla, Calif.). Hybridization was performed according to the membrane
manufacturer’s recommendations with random hexamer-primed
DNA probes and a final washing with 1? SSPE–0.5% sodium dodecyl sulfate at
37°C. Autoradiography was performed with either a Molecular Dynamics Phos-
phorImager (Amersham, Arlington Heights, Ill.) or conventional X-ray film. The
HES1 probe was described previously (36). The GAPDH probe was a 168-bp
fragment spanning bases 201 to 368 (GenBank M32599) derived by PCR from a
mouse pre-B cell cDNA library and cloned into pKS? (Stratagene).
Protein purification. Bacterial expression plasmids encoding MAML1 or
NOTCH1 polypeptides were transformed into BL21(DE3) or BL21(DE3)pLysS
Escherichia coli (Invitrogen). Cultures grown in Luria-Bertani broth were in-
duced with 1 mM isopropyl-[exists]-D-thiogalactopyranoside at 37°C for 3 h.
After collection by centrifugation, bacterial pellets were resuspended in 1/50th of
the original culture volume of a solution containing ice-cold PBS, pH 7.4, con-
taining 5 mM ?-mercaptoethanol, 2 ?g of aprotinin/ml, 1 ?g of leupeptin/ml, and
0.5 mM phenylmethylsulfonyl fluoride. Bacteria were lysed by three cycles of
freezing and thawing, each followed by sonication for 90 s (3 times for 30 s each)
on ice with a Branson Sonifier (Branson Ultrasonics, Danbury, Conn.).
MAML1 peptides were prepared from inclusion bodies as follows. Triton
X-100 (1% [vol/vol]) was added to the lysates, which were mixed for 60 min at
25°C. Inclusion bodies were collected by centrifugation and were solubilized in
PBS containing 6 M guanidine-HCl and 5 mM ?-mercaptoethanol overnight at
25°C. After removal of insoluble material by centrifugation, soluble proteins
were applied to an Ni-nitrilotriacetic acid agarose (Qiagen, Valencia, Calif.)
column (bed volume of ?1/300th of the initial culture volume). After extensive
washing, bound proteins were step eluted with PBS solutions containing 6 M
guanidine-HCl and 5 mM ?-mercaptoethanol equilibrated to pH 6.3, 5.9, and 4.5
and were analyzed by electrophoresis in tricine gels (48). Fractions containing
six-His–MAML1 peptides were pooled and adjusted to pH 7.4, reduced by
addition of 10 mM dithiothreitol, and dialyzed at 4°C over several days in
Spectra/Por membranes (Spectrum Laboratories, Santa Dominguez, Calif.)
against PBS, pH 7.4, containing 5 mM ?-mercaptoethanol and decreasing con-
centrations of guanidine-HCl. A precipitate enriched for MAML1 peptides
formed during removal of guanidine-HCl that was collected by centrifugation,
solubilized in 5% (vol/vol) acetic acid, and freeze dried. Lyophilized peptides
were dissolved in 50 mM piperazine sulfonic acid, pH 6.2, containing 0.5 mM
EDTA and 1 mM dithiothreitol, and were incubated overnight at 25°C with
recombinant TEV protease (Invitrogen). After dialysis against 50 mM Tris, pH
6.0, containing 0.5 mM EDTA and 5 mM ?-mercaptoethanol, the cleaved pep-
tides were acidified by addition of 5% (vol/vol) acetic acid and were applied to
a C18 preparative-scale high-performance liquid chromatography column, which
was developed with a 20 to 40% acetonitrile gradient. Fractions containing
eluted peptides (assessed by monitoring optical density at 209 nm) were col-
lected, lyophilized, resuspended in acidified H2O (pH 5.5), and stored at ?80°C.
Fractions containing purified MAML1 peptides (as judged by the presence of a
single species of appropriate size in tricine gels) were used in electrophoretic
mobility shift assays (EMSA).
NOTCH1 polypeptides were purified from bacterial lysates as described pre-
viously (60). Lysates containing soluble glutathione S-transferase-NOTCH1
polypeptides were incubated for 4 h at 4°C with glutathione Sepharose 4B beads
(Pharmacia). After extensive washing, the beads were incubated overnight at
25°C in 50 mM Tris, pH 8.0, containing 0.5 mM EDTA, 1 mM dithiothreitol, and
recombinant TEV protease. NOTCH1 polypeptides were purified to electro-
phoretic homogeneity by ion exchange chromatography on Mono-Q resin (Am-
ersham) followed by gel filtration on a Superdex 200 column (Amersham).
CSL was immunopurified from transiently overexpressing 293T cells by using
a monoclonal anti-myc antibody (clone 9E10, kindly provided by Jeffrey Parvin)
followed by elution with myc peptide (Research Genetics), as described previ-
EMSA. Conditions for EMSA were as described previously (60). Briefly, pro-
tein complexes were allowed to form for 30 min at 30°C in a 15-?l volume
containing 104cpm of end-labeled probe (23), 1 ?l of immunopurified CSL, 50
ng of ICN1 polypeptides, and/or 10 to 50 ng of MAML1 peptides. Electrophore-
sis was performed at 175 V in a 10% Tris-glycine-EDTA gel, which was dried and
analyzed by autoradiography.
Comparison of presenilin inhibitors in reporter gene assays.
Small-molecule peptidomimetic inhibitors of presenilins block
proteolysis and nuclear translocation of membrane-tethered
?E (5). We used transient expression assays to compare the
VOL. 23, 2003 NOTCH SIGNALS IN T-ALL657
effects of 12 such inhibitors on ?E-dependent transactivation
of CSL. Our expression construct encodes a form of ?E con-
sisting of the endogenous NOTCH1 signal peptide fused to the
NTM subunit at a position 61 residues external to the trans-
membrane domain. ?E thus requires sequential proteolytic
cleavage by metalloprotease (S2) and presenilin (S3) for nu-
clear access (see Fig. 1a). The tested compounds vary widely in
inhibitory potency (Fig. 2). The potency of any individual com-
pound in inhibiting ?E transactivation is highly correlated with
its ability to inhibit ?-amyloid precursor protein cleavage (M.
Wolfe, unpublished data).
?E-induced T-ALL cell growth is suppressed by presenilin
inhibitors. DFP-AA, a potent inhibitor of ?E-dependent
transactivation (Fig. 2), was assessed for its effects on the
growth of T6E cells, a murine T-ALL cell line derived from a
?E-induced tumor (40). DFP-AA causes significant dose-de-
pendent suppression of T6E growth at concentrations as low as
100 nM (Fig. 3, left panel). For three other compounds, WPE
III-18, MW III-36A, and WPE III-109 (strong, moderate, and
negligible inhibitors of ?E transactivation, respectively; Fig. 2),
growth-suppressive activity also correlates with ?E inhibition
(data not shown).
To control for NOTCH-independent effects of the inhibi-
tors, we also tested DFP-AA on I22 cells, a murine T-ALL cell
line derived from an ICN1-induced tumor (40). At high doses,
DFP-AA produces a small decrement in I22 growth, suggesting
that any NOTCH-independent effects are minimal (Fig. 3,
Presenilin inhibitors perturb cell cycle progression and in-
duce apoptosis of T6E cells. T6E cells demonstrate a dose-
dependent increase in G1/G0fraction and a decrease in S-
phase fraction after only 3 days of treatment with DFP-AA,
while I22 cells are unaffected by up to 8 days of treatment (Fig.
4a and b). After 8 days, T6E, but not I22, cultures also show a
significant accumulation of dead or dying cells, which is re-
flected by the presence of a large cell fraction with sub-G1
DNA content (Fig. 4c). This is accompanied by dose-depen-
dent induction of apoptosis in T6E, but not I22, cells as judged
by an increase in Annexin-V?/7-AAD?cells (Fig. 4d). These
data indicate ?E-expressing T-ALL cells selectively demon-
strate altered cell cycle progression and apoptosis in response
to DFP-AA treatment.
Transforming alleles of ICN1 rescue ?E-expressing T-ALL
cells from presenilin inhibition. To confirm that the growth
suppression of ?E-expressing cells results from inhibition of
NOTCH signaling, we tested whether ICN1 isoforms that do
not require presenilin cleavage for nuclear access (ICN1,
?RAM?P, and ?TAD?P; Fig. 1a) prevent presenilin inhibi-
tor-mediated growth suppression. T6E cells were transduced
with various ICN1 isoforms along with a marker, GFP, into
T6E cells and then were treated with DFP-AA. Retroviral
titers were adjusted so that only a subpopulation of cells would
be transduced (?10 to 20% GFP?), allowing the growth of
FIG. 2. Potency of presenilin inhibitor compounds in suppression
of transcriptional stimulation by ?E. U2OS cells were transiently
transfected with a ?E expression construct along with a CSL-luciferase
reporter. Average normalized luciferase reporter activity (? standard
deviations) from triplicate samples is expressed as a percentage of that
observed in mock-treated cells.
FIG. 3. Suppression of T6E cell growth by the presenilin inhibitor compound DFP-AA. T6E (left panel) and I22 (right panel) cells were
cultured in the presence of the indicated concentrations of DFP-AA or carrier (mock) and were counted daily. Extrapolated cell counts were
calculated as described in Materials and Methods.
658WENG ET AL.MOL. CELL. BIOL.
transduced and nontransduced cells to be compared under
identical culture conditions. As depicted in Fig. 5a, ICN1 and
?RAM?P, but not ?TAD?P, rescue T6E cells only under
conditions of DFP-AA treatment; in mock-treated cells, these
isoforms have no effect. Additionally, ICN1, but not ?TAD?P,
prevents the drug-induced increase in G1/G0fraction (Fig. 5b),
while ?RAM?P shows an intermediate phenotype. Interest-
ingly, the in vitro phenotypes of these three Notch alleles
correlate well with their relative transforming potentials in a
murine T-ALL model (4). Specifically, ICN1 induces T-ALL
rapidly and activates CSL-dependent transcription strongly,
?RAM?P induces T-ALL more slowly and activates CSL-
dependent transcription moderately, and ?TAD?P is nontu-
morigenic and activates CSL-dependent transcription only
weakly. These data show that the effects of presenilin inhibitors
are likely mediated through inhibition of ICN1 production and
that nuclear access by the ANK and C-terminal TADs of ICN1
is sufficient (and the TAD is necessary) for continued cell
Presenilin inhibitors result in accumulation of a stable
NOTCH1 processing intermediate and downregulate HES1
transcription. Although proteolytic products derived from ?E
in T6E cells are not detected under normal circumstances, we
suspected inhibition of presenilin activity might cause accumu-
lation of ?E* (Fig. 1a), the product of metalloprotease cleav-
age of ?E. Indeed, treatment of T6E cells with DFP-AA per-
mits detection of a new NOTCH1 species of the expected size
of ?E* (Fig. 6a). This polypeptide is first detected after 3 h of
treatment and continues to accumulate during the time course
of the experiment, suggesting that ?E* is fairly stable when
presenilin is inhibited. No change in NOTCH1 polypeptides
occurred in I22 cells treated with DFP-AA or T6E cells treated
with dimethyl sulfoxide carrier alone.
To link DFP-AA treatment effects to known NOTCH1 sig-
naling events, we assessed its effects on a well-characterized
NOTCH1/CSL target, HES1. HES1 mRNA transcripts de-
crease rapidly in T6E cells treated with DFP-AA, falling to
undetectable levels within 6 h (Fig. 6b). In contrast, DFP-AA
FIG. 4. Presenilin inhibitor treatment alters cell cycle progression and induces apoptosis in T6E, but not I22, cells. (a and b) Cell cycle analysis.
T6E and I22 cells were treated with the indicated concentrations of DFP-AA for 3 and 8 days, respectively. DNA content was measured by flow
cytometry after staining with propidium iodide. (a) Representative propidium iodide (PI) fluorescence histograms with superimposed cell cycle
analysis models (including background correction). (b) Cell cycle fractions determined from histograms as for panel a. (c and d) Apoptosis analysis.
(c) Sub-G1fractions determined by using histograms as for panel a. (d) T6E and I22 cells treated with DFP-AA for 8 days were dual-stained with
Annexin-V (for apoptotic cells) and 7-ADD (to exclude dead cells) and were analyzed by flow cytometry. Percentages of apoptotic cells
(Annexin-V?/7-AAD?) are indicated after excluding cell debris by forward/side scatter gating.
VOL. 23, 2003 NOTCH SIGNALS IN T-ALL659
has no effect on HES1 expression in I22 cells. These data
confirm that DFP-AA treatment inhibits presenilin-dependent
cleavage of ?E in T6E cells and strongly suggest small amounts
of ?E-derived, short-lived ICN1 are essential for maintenance
of T6E cell growth and survival.
Mapping and characterization of dominant-negative MAML1
peptides. Recent data suggest that ICN association with CSL is
necessary for loading of MAML1 (60) and that both ICN and
MAML1 are essential for activation of CSL-dependent tran-
scription (20). Consistent with this view, truncated forms of
MAML1 retaining an N-terminal ICN/CSL interaction domain
but lacking a C-terminal TAD act as dominant-negative inhib-
itors of ICN function (20, 60).
To test the idea that dominant-negative forms of MAML1
might be general inhibitors of NOTCH1-transformed T-ALL
cell growth, we first determined the minimal portion of
MAML1 needed for ternary complex formation in an EMSA.
As part of a parallel study, we also defined a portion of ICN1
spanning the RAM and ANK domains as the minimal domain
of NOTCH1 that is needed for stable ternary complex forma-
tion in vitro (Y. Nam, unpublished data). This RAM-ANK
polypeptide, full-sized CSL, and MAML1 residues 13 to 74
[termed MAML1(13-74)] were sufficient for ternary complex
formation on DNA (Fig. 7a), whereas shorter MAML1 pep-
tides (residues 22 to 74, 13 to 63, and 13 to 52) associated
weakly with CSL/RAM-ANK. MAML1(13-74) also defined
FIG. 5. Leukemogenic forms of ICN1 rescue T6E cells from presenilin inhibitor-mediated growth suppression. (a) Growth advantage of
ICN1-transduced cells under conditions of presenilin inhibitor treatment. T6E cells were transduced with retroviruses encoding various consti-
tutively nuclear forms of NOTCH1 (shown in Fig. 1a) and GFP on a bicistronic mRNA or GFP alone and then were treated with 1 ?M DFP-AA
or carrier (mock) beginning at day 3 posttransduction and continuing for the duration of the experiment. The percentage of GFP?cells in each
of the cultures was determined daily by flow cytometry, gating for live cells by forward/side scatter criteria. All cultures were repeated in duplicate;
a single representative experiment is shown. (b) Cell cycle analysis. T6E cells from the experiment depicted in panel a were harvested after 9 days
of exposure to 1 ?M DFP-AA or carrier (mock) and were stained with DRAQ5 dye. DNA content was measured by DRAQ5 fluorescence in GFP?
and GFP?subpopulations by flow cytometry.
660WENG ET AL.MOL. CELL. BIOL.
the minimal domain necessary for strong dominant-negative
activity in ICN1 gene reporter assays (Fig. 7b). These results
indicate that MAML1(13-74) is sufficient for formation of sta-
ble, transcriptionally inert ICN1/CSL/MAML1 ternary com-
Dominant-negative MAML1 peptides suppress the growth
of NOTCH1-transformed T-ALL cell lines. To create a readily
detectable form of dominant-negative MAML1, we fused the
minimal MAML1 dominant-negative peptide [MAML1(13-
74)] to GFP. In pilot experiments, MAML1(13-74)-GFP fusion
protein expressed from the MSCV retroviral long terminal
repeat promoter retained strong dominant-negative activity in
reporter gene assays (data not shown).
Various lymphoid cell lines were then transduced with dom-
inant-negative MAML1(13-74)-GFP or control GFP retrovi-
ruses. In initial experiments, growth of transduced (GFP?) and
untransduced (GFP?) cell fractions was compared within un-
sorted cultures. Growth suppression of the transduced popu-
lation would thus lead to a decreasing GFP?percentage over
time. As depicted in Fig. 8a, transduction with dominant-neg-
ative MAML1(13-74)-GFP virus caused significant growth sup-
pression of murine T6E and I22 cell lines as well as SUP-T1, a
human T-ALL cell line with a chromosomal translocation in-
volving NOTCH1 that leads to expression of ICN1-like
polypeptides (2). Transduction with GFP-only virus did not
inhibit the growth of any of these lines. Human BJAB cells and
murine BW5147 cells, both harboring apparently normal
NOTCH1 alleles (data not shown), were unaffected by trans-
duction with MAML1(13-74)-GFP virus (Fig. 8a), suggesting
the growth-suppressive effects of dominant-negative MAML1
are limited to NOTCH1-transformed cell lines.
To define the growth-suppressive effects of MAML1(13-74)-
GFP virus further, we measured absolute growth rates and
performed cell cycle analysis on SUP-T1 and BW5147 cultures
that contained ?95% retrovirally transduced, GFP?cells. Hu-
man SUP-T1 cells transduced with MAML1(13-74)-GFP virus
showed a decreased absolute growth rate (Fig. 8b), an in-
creased G1/G0fraction, and a decreased S-phase fraction (Fig.
8c) compared to those of GFP-only control cells, whereas no
difference was seen with BW5147 cells.
These studies provide strong evidence that NOTCH1-in-
duced T-ALLs require persistent NOTCH1 signaling for
growth and survival. NOTCH signaling was inhibited at two
distinct steps. In one set of experiments, presenilin inhibitors
prevented cleavage and subsequent nuclear translocation of
ICN, leading to growth inhibition and death of a ?E-expressing
murine T-ALL cell line. In a second set of experiments,
MAML1 dominant-negative peptides were used to inhibit nu-
clear NOTCH1, causing growth inhibition and death of
Notch1-induced human and murine T-ALL cell lines. To-
gether these findings show that signals transduced by nuclear
NOTCH1 are required for growth and survival of Notch1-
transformed pre-T cells.
FIG. 6. Presenilin inhibitor treatment blocks NOTCH1 signal
transduction in T6E, but not I22, cells. (a) Effects on NOTCH1
polypeptides. T6E and I22 cells were treated with 1 ?M DFP-AA for
the indicated periods of time. NOTCH1 polypeptides were then im-
munoprecipitated from whole-cell extracts with an antibody directed
against the intracellular domain of NOTCH1 (?-TC) and were ana-
lyzed on a Western blot stained with ?-TC. ?E, ?E*, and ICN are
NOTCH1-derived polypeptides diagrammed in Fig. 1. (b) Effects on
HES1 transcript level. After T6E and I22 cells were treated with 1 ?M
DFP-AA for the indicated periods of time, total RNA was collected
and analyzed on a Northern blot with probes for HES1 and GAPDH.
FIG. 7. Mapping and functional characterization of dominant-neg-
ative MAML1 peptides. (a) Mapping MAML1 residues needed for
ternary complex formation. Highly purified MAML1 peptides incu-
bated with recombinant ICN1 RAM-ANK and immunopurified CSL
were scored for complex formation in an EMSA. MAML1 peptides
were included at increasing concentrations of up to a fivefold molar
excess over the concentration of RAM-ANK. (b) Dominant-negative
activities of MAML1 peptides. Each well of a 24-well plate containing
U2OS cells was transiently transfected with pcDNA3 expression con-
structs for ICN1 (10 ng), various MAML1 peptides (50 ng), a CSL-
luciferase reporter (125 ng), and a Renilla luciferase internal control
reporter (2.5 ng). Mean normalized luciferase activity (? standard
deviations) from triplicate wells is expressed relative to that observed
in cells transfected with reporter constructs plus empty pcDNA3 (60
VOL. 23, 2003 NOTCH SIGNALS IN T-ALL661
The observation that ?E proteolysis and nuclear transloca-
tion are required for proliferation and survival of T6E cells has
several implications. ?E-expressing T6E cells have levels of
nuclear ICN1 that are below the sensitivities of standard anti-
body-based detection methods (40). Nevertheless, ?E is a po-
tent activator of CSL-dependent reporter genes (3) and an
inducer of T-ALL in our murine model (40), apparently be-
cause presenilin-dependent processing leads to inappropriately
high levels of nuclear ICN1. In this regard it is noteworthy that,
despite convincing evidence that nuclear access is essential for
NOTCH function (25, 49, 52, 53), it is difficult or impossible to
detect NOTCH in the nucleus of normal cells. It follows that
subtle increases in nuclear levels of NOTCH could have an
important impact on the behavior of malignant cells.
Other observations also provide support for this possibility.
Enforced expression of the NOTCH ligand DELTA-LIKE-4 in
bone marrow cells produces T-ALL in mice (61), implying that
transformation can occur merely through inappropriate acti-
vation of otherwise normal NOTCH receptors. Ligand-medi-
ated NOTCH signaling also stimulates the growth of lymphoid
cell lines derived from classical Hodgkin’s lymphoma and ana-
plastic large-cell lymphoma (30). Many mutations producing
gain-of-function phenotypes in invertebrates consist of single
amino acid substitutions within extracellular portions of
NOTCH receptors (11, 22, 34). In each case, relatively small
increases in nuclear NOTCH arising through metalloprotease-
and presenilin-mediated proteolytic cleavages (akin to the sit-
uation in ?E-induced T-ALLs) likely produce the observed
phenotypes. Presenilin inhibitors may be useful in screening
cell lines and primary tumors for evidence of ongoing NOTCH
processing (on the basis of accumulation of ?E*, as depicted in
Fig. 6a) and dependence on NOTCH nuclear access for growth
FIG. 8. Dominant-negative MAML1 peptides specifically suppress growth of human and murine NOTCH1-transformed T-cell lines. (a)
Growth suppression of cells expressing the dominant-negative MAML1(13-74)-GFP fusion protein. Each cell line was transduced with
MAML1(13-74)-GFP or GFP retrovirus at titers such that only a subpopulation of cells (?40 to 70%) were transduced. The percentage of GFP?
cells was determined daily by flow cytometry, gating for live cells by forward/side scatter criteria. The percentage of GFP?cells remaining at each
day is expressed as a fraction of the initial (day 3 posttransduction) GFP?percentage. All cell lines except BJAB were used in three independent
experiments; a single representative experiment is shown. (b) Absolute growth rates of SUPT-T1 and BW5147 cultures in which ?95% of cells had
been transduced by the indicated retroviruses. Cell counts were performed daily starting at day 2 postretroviral transduction, and extrapolated cell
counts were calculated as described in Materials and Methods. (c) Cell cycle effects of MAML1(13-74)-GFP. DNA content was measured from
the SUP-T1 and BW5147 cultures depicted in panel b on day 7 posttransduction (corresponding to day 5 in panel b). Cells were stained with
propidium iodide and were analyzed by flow cytometry. MamGFP, MAML1(13-74)-GFP; GFP, GFP only.
662WENG ET AL.MOL. CELL. BIOL.
and survival (58), using the strategy of ICN rescue to control
for NOTCH-independent effects. Moreover, NOTCH signals
can also transform primary baby hamster kidney cells (9) and
murine mammary epithelial cells (46), indicating that screens
for Notch activity will have utility in nonhematopoietic tumors
The requirement for very low levels of nuclear ICN1 in
?E-expressing T6E cells also points out an incongruity in
NOTCH signaling relevant to transformation and develop-
ment. Expression constructs encoding ?E and ICN1 are equi-
potent inducers of T-ALL in our murine model, despite large
differences in the levels of nuclear ICN1 produced by these two
alleles (40). Furthermore, T-ALLs arising from bone marrow
cells transduced with ICN1 alleles almost uniformly arise from
a GFP-bright, ICN1hicell population (4), suggesting that high
levels of engineered ICN1 are required for efficient transfor-
mation. A similar paradox has been observed in the developing
Drosophila eye, where ?E causes more pronounced pheno-
types than ICN despite the presence of substantially more
nuclear NOTCH in ICN-expressing cells (19). These data sug-
gest ?E-derived forms of ICN are more potent on a molecule-
for-molecule basis in activating downstream signals. Conceiv-
ably, during or subsequent to proteolysis ?E might undergo
phosphorylation or other modifying events that augment
downstream signaling with greater efficiency than engineered
ICN polypeptides. One such modifier could be SEL-5, a serine/
threonine kinase that acts upstream of nuclear events to en-
hance NOTCH signaling in C. elegans (18).
Once ICN1 reaches the nucleus, recruitment of coactivators
is important for strong activation of CSL and T-cell transfor-
mation. By using a murine model of leukemogenesis, it was
previously noted that the minimal transforming portion of
ICN1 includes the ANK domain and a C-terminal TAD (4).
The same portion of ICN1 is sufficient to rescue T-ALL cells
from presenilin inhibition, suggesting that the signals required
for growth ex vivo are similar or identical to those required for
transformation in vivo.
Likely roles for the ANK domain, which is required for all
NOTCH functions, are to form weak contacts with CSL (3, 31,
54) and to recruit MAML1 (60). A critical role for MAML1
binding is supported by growth suppression of NOTCH1-in-
duced T-ALLs by dominant-negative MAML1, a potent inhib-
itor of CSL activation. These effects could indicate either that
MAML recruitment is critical for CSL activation or that mul-
tiple, different coactivators are loaded onto ICN through the
same contact site. In support of the former possibility, recent
work using an in vitro transcription system showed that ICN1
activation of CSL-dependent transcription requires MAML1
(20), which may serve as a docking site for p300 (20) and other
uncharacterized coactivators (60). Activation of CSL in vitro
also appears to require the ICN1 TAD (20). This same domain
is necessary for strong CSL activation in reporter gene assays
(4, 32, 33) and may serve to recruit a different class of coacti-
vators (32). Thus, CSL/ICN1/MAML1 is likely an essential
subcomplex within a larger multiprotein assembly. In accor-
dance with this prediction, Capobianco’s group recently iden-
tified and partially purified CSL, ICN1, and MAML1 together
in a ?1.5-MDa complex in SUP-T1 cells (28).
Our findings demonstrate that NOTCH signaling influences
the growth potential of transformed T-ALL cells directly, in-
dicating that the mechanism of transformation extends beyond
effects on differentiation. NOTCH1 signaling inhibitors sup-
press the growth of NOTCH1-induced T-ALL lines by per-
NOTCH1 was shown previously to upregulate cyclin D1 in
BHK cells (47) and to rescue T-cell lines from glucocorticoid-
induced apoptosis (13). It will be of interest to determine the
molecular mechanisms underlying growth suppression and ap-
optosis in NOTCH1-transformed T cells in which both nuclear
translocation of ICN1 and recruitment of coactivators are im-
portant for transduction of growth and survival signals. Thus,
these studies provide proof of principle for the targeting of
nuclear Notch complexes in the treatment of NOTCH-induced
T-ALL. Furthermore, the Notch-specific inhibitors described
herein may prove useful in determining the importance of
NOTCH signaling in other forms of T-ALL and in other can-
cers as well.
A.P.W. was supported by an NIH postdoctoral training grant. This
work was also supported by Public Health Service grants to J.C.A.,
S.C.B., J.G., and W.S.P. from the National Cancer Institute. W.S.P. is
a Scholar of the Leukemia and Lymphoma Society.
1. Artavanis-Tsakonas, S., M. D. Rand, and R. J. Lake. 1999. Notch signaling:
cell fate control and signal integration in development. Science 284:770–776.
2. Aster, J., W. Pear, R. Hasserjian, H. Erba, F. Davi, B. Luo, M. Scott, D.
Baltimore, and J. Sklar. 1994. Functional analysis of the TAN-1 gene, a
human homolog of Drosophila notch. Cold Spring Harbor Symp. Quant.
3. Aster, J. C., E. S. Robertson, R. P. Hasserjian, J. R. Turner, E. Kieff, and J.
Sklar. 1997. Oncogenic forms of NOTCH1 lacking either the primary bind-
ing site for RBP-J? or nuclear localization sequences retain the ability to
associate with RBP-J? and activate transcription. J. Biol. Chem. 272:11336–
4. Aster, J. C., L. Xu, F. G. Karnell, V. Patriub, J. C. Pui, and W. S. Pear. 2000.
Essential roles for ankyrin repeat and transactivation domains in induction
of T-cell leukemia by Notch1. Mol. Cell. Biol. 20:7505–7515.
5. Berezovska, O., C. Jack, P. McLean, J. C. Aster, C. Hicks, W. Xia, M. S.
Wolfe, W. T. Kimberly, G. Weinmaster, D. J. Selkoe, and B. T. Hyman. 2000.
Aspartate mutations in presenilin and gamma-secretase inhibitors both im-
pair notch1 proteolysis and nuclear translocation with relative preservation
of notch1 signaling. J. Neurochem. 75:583–593.
6. Berry, L. W., B. Westlund, and T. Schedl. 1997. Germ-line tumor formation
caused by activation of glp-1, a Caenorhabditis elegans member of the Notch
family of receptors. Development 124:925–936.
7. Brou, C., F. Logeat, N. Gupta, C. Bessia, O. LeBail, J. R. Doedens, A.
Cumano, P. Roux, R. A. Black, and A. Israel. 2000. A novel proteolytic
cleavage involved in Notch signaling: the role of the disintegrin-metallopro-
tease TACE. Mol. Cell 5:207–216.
8. Bush, G., G. diSibio, A. Miyamoto, J. B. Denault, R. Leduc, and G. Wein-
master. 2001. Ligand-induced signaling in the absence of furin processing of
Notch1. Dev. Biol. 229:494–502.
9. Capobianco, A. J., P. Zagouras, C. M. Blaumueller, S. Artavanis-Tsakonas,
and J. M. Bishop. 1997. Neoplastic transformation by truncated alleles of
human NOTCH1/TAN1 and NOTCH2. Mol. Cell. Biol. 17:6265–6273.
10. Carlesso, N., J. C. Aster, J. Sklar, and D. T. Scadden. 1999. Notch1-induced
delay of human hematopoietic progenitor cell differentiation is associated
with altered cell cycle kinetics. Blood 93:838–848.
11. De Celis, J. F., and S. J. Bray. 2000. The Abruptex domain of Notch
regulates negative interactions between Notch, its ligands and Fringe. De-
12. Deftos, M. L., Y. W. He, E. W. Ojala, and M. J. Bevan. 1998. Correlating
notch signaling with thymocyte maturation. Immunity 9:777–786.
13. De Strooper, B., W. Annaert, P. Cupers, P. Saftig, K. Craessaerts, J. S.
Mumm, E. H. Schroeter, V. Schruvers, M. S. Wolfe, W. J. Ray, A. Goate, and
R. Kopan. 1999. A presenilin-1-dependent-secretase-like protease mediates
release of Notch intracellular domain. Nature 398:518–522.
14. Dovey, H. F., V. John, J. P. Anderson, L. Z. Chen, P. de Saint Andrieu, L. Y.
Fang, S. B. Freedman, B. Folmer, E. Goldbach, E. J. Holsztynska, K. L. Hu,
K. L. Johnson-Wood, S. L. Kennedy, D. Kholodenko, J. E. Knops, L. H.
Latimer, M. Lee, Z. Liao, I. M. Lieberburg, R. N. Motter, L. C. Mutter, J.
VOL. 23, 2003 NOTCH SIGNALS IN T-ALL663
Nietz, K. P. Quinn, K. L. Sacchi, P. A. Seubert, G. M. Shopp, E. D. Thorsett, Download full-text
J. S. Tung, J. Wu, S. Yang, C. T. Yin, D. B. Schenk, P. C. May, L. D. Altstiel,
M. H. Bender, L. N. Boggs, T. C. Britton, J. C. Clemens, D. L. Czilli, D. K.
Dieckman-McGinty, J. J. Droste, K. S. Fuson, B. D. Gitter, P. A. Hyslop,
E. M. Johnstone, W. Y. Li, S. P. Little, T. E. Mabry, F. D. Miller, and J. E.
Audia. 2001. Functional gamma-secretase inhibitors reduce beta-amyloid
peptide levels in brain. J. Neurochem. 76:173–181.
15. Doyle, T. G., C. Wen, and I. Greenwald. 2000. SEL-8, a nuclear protein
required for LIN-12 and GLP-1 signaling in Caenorhabditis elegans. Proc.
Natl. Acad. Sci. USA 97:7877–7881.
16. Ellisen, L. W., J. Bird, D. C. West, A. L. Soreng, T. C. Reynolds, S. D. Smith,
and J. Sklar. 1991. TAN-1, the human homolog of the Drosophila notch
gene, is broken by chromosomal translocations in T lymphoblastic neo-
plasms. Cell 66:649–661.
17. Esler, W. P., W. T. Kimberly, B. L. Ostaszewski, W. Ye, T. S. Diehl, D. J.
Selkoe, and M. S. Wolfe. 2002. Activity-dependent isolation of the presenilin-
gamma-secretase complex reveals nicastrin and a gamma substrate. Proc.
Natl. Acad. Sci. USA 99:2720–2725.
18. Fares, H., and I. Greenwald. 1999. SEL-5, a serine/threonine kinase that
facilitates lin-12 activity in Caenorhabditis elegans. Genetics 153:1641–1654.
19. Fortini, M. E., I. Rebay, L. A. Caron, and S. Artavanis-Tsakonas. 1993. An
activated Notch receptor blocks cell-fate commitment in the developing
Drosophila eye. Nature 365:555–557.
20. Fryer, C. J., E. Lamar, I. Turbachova, C. Kintner, and K. A. Jones. 2002.
Mastermind mediates chromatin-specific transcription and turnover of the
Notch enhancer complex. Genes Dev. 16:1397–1411.
21. Go, M. J., D. S. Eastman, and S. Artavanis-Tsakonas. 1998. Cell prolifera-
tion control by Notch signaling in Drosophila development. Development
22. Greenwald, I., and G. Seydoux. 1990. Analysis of gain-of-function mutations
of the lin-12 gene of Caenorhabditis elegans. Nature 346:197–199.
23. Henkel, T., P. D. Ling, S. D. Hayward, and M. G. Peterson. 1994. Mediation
of Epstein-Barr virus EBNA2 transactivation by recombination signal-bind-
ing protein J?. Science 265:92–95.
24. Hsieh, J. J., T. Henkel, P. Salmon, E. Robey, M. G. Peterson, and S. D.
Hayward. 1996. Truncated mammalian Notch1 activates CBF1/RBPJ?-re-
pressed genes by a mechanism resembling that of Epstein-Barr virus
EBNA2. Mol. Cell. Biol. 16:952–959.
25. Huppert, S. S., A. Le, E. H. Schroeter, J. S. Mumm, M. T. Saxena, L. A.
Milner, and R. Kopan. 2000. Embryonic lethality in mice homozygous for a
processing-deficient allele of Notch1. Nature 405:966–970.
26. Izon, D. J., J. A. Punt, L. Xu, F. G. Karnell, D. Allman, P. S. Myung, N. J.
Boerth, J. C. Pui, G. A. Koretzky, and W. S. Pear. 2001. Notch1 regulates
maturation of CD4? and CD8? thymocytes by modulating TCR signal
strength. Immunity 14:253–264.
27. Jeffries, S., and A. J. Capobianco. 2000. Neoplastic transformation by Notch
requires nuclear localization. Mol. Cell. Biol. 20:3928–3941.
28. Jeffries, S., D. J. Robbins, and A. J. Capobianco. 2002. Characterization of a
high-molecular-weight Notch complex in the nucleus of Notch(ic)-trans-
formed RKE cells and in a human T-cell leukemia cell line. Mol. Cell. Biol.
29. Jehn, B. M., W. Bielke, W. S. Pear, and B. A. Osborne. 1999. Protective
effects of notch-1 on TCR-induced apoptosis. J. Immunol. 162:635–638.
30. Jundt, F., I. Anagnostopoulos, R. Forster, S. Mathas, H. Stein, and B.
Dorken. 2002. Activated Notch1 signaling promotes tumor cell proliferation
and survival in Hodgkin and anaplastic large cell lymphoma. Blood 99:3398–
31. Kato, H., Y. Taniguchi, H. Kurooka, S. Minoguchi, T. Sakai, S. Nomura-
Okazaki, K. Tamura, and T. Honjo. 1997. Involvement of RBP-J in biolog-
ical functions of mouse Notch1 and its derivatives. Development 124:4133–
32. Kurooka, H., and T. Honjo. 2000. Functional interaction between the mouse
notch1 intracellular region and histone acetyltransferases PCAF and GCN5.
J. Biol. Chem. 275:17211–17220.
33. Kurooka, H., K. Kuroda, and T. Honjo. 1998. Roles of the ankyrin repeats
and C-terminal region of the mouse notch1 intracellular region. Nucleic
Acids Res. 26:5448–5455.
34. Lieber, T., S. Kidd, E. Alcamo, V. Corbin, and M. W. Young. 1993. Antineu-
rogenic phenotypes induced by truncated Notch proteins indicate a role in
signal transduction and may point to a novel function for Notch in nuclei.
Genes Dev. 7:1949–1965.
35. Logeat, F., C. Bessia, C. Brou, O. LeBail, S. Jarriault, N. G. Seidah, and A.
Israel. 1998. The Notch1 receptor is cleaved constitutively by a furin-like
convertase. Proc. Natl. Acad. Sci. USA 95:8108–8112.
36. Luo, B., J. C. Aster, R. P. Hasserjian, F. Kuo, and J. Sklar. 1997. Isolation
and functional analysis of a cDNA for human Jagged2, a gene encoding a
ligand for the Notch1 receptor. Mol. Cell. Biol. 17:6057–6067.
37. Matsuno, K., M. J. Go, X. Sun, D. S. Eastman, and S. Artavanis-Tsakonas.
1997. Suppressor of Hairless-independent events in Notch signaling imply
novel pathway elements. Development 124:4265–4273.
38. Mumm, J. S., and R. Kopan. 2000. Notch signaling: from the outside in. Dev.
39. Nofziger, D., A. Miyamoto, K. M. Lyons, and G. Weinmaster. 1999. Notch
signaling imposes two distinct blocks in the differentiation of C2C12 myo-
blasts. Development 126:1689–1702.
40. Pear, W. S., J. C. Aster, M. L. Scott, R. P. Hasserjian, B. Soffer, J. Sklar, and
D. Baltimore. 1996. Exclusive development of T cell neoplasms in mice
transplanted with bone marrow expressing activated Notch alleles. J. Exp.
41. Petcherski, A. G., and J. Kimble. 2000. LAG-3 is a putative transcriptional
activator in the C. elegans Notch pathway. Nature 405:364–368.
42. Pui, J. C., D. Allman, L. Xu, S. DeRocco, F. G. Karnell, S. Bakkour, J. Y. Lee,
T. Kadesch, R. R. Hardy, J. C. Aster, and W. S. Pear. 1999. Notch1 expres-
sion in early lymphopoiesis influences B versus T lineage determination.
43. Radtke, F., A. Wilson, G. Stark, M. Bauer, J. van Meerwijk, H. R. Mac-
Donald, and M. Aguet. 1999. Deficient T cell fate specification in mice with
an induced inactivation of Notch1. Immunity 10:547–558.
44. Ramain, P., K. Khechumian, L. Seugnet, N. Arbogast, C. Ackermann, and P.
Heitzler. 2001. Novel Notch alleles reveal a Deltex-dependent pathway re-
pressing neural fate. Curr. Biol. 11:1729–1738.
45. Rebay, I., R. J. Fleming, R. G. Fehon, L. Cherbas, P. Cherbas, and S.
Artavanis-Tsakonas. 1991. Specific EGF repeats of Notch mediate interac-
tions with Delta and Serrate: implications for Notch as a multifunctional
receptor. Cell 67:687–699.
46. Robbins, J., B. J. Blondel, D. Gallahan, and R. Callahan. 1992. Mouse
mammary tumor gene int-3: a member of the notch gene family transforms
mammary epithelial cells. J. Virol. 66:2594–2599.
47. Ronchini, C., and A. J. Capobianco. 2001. Induction of cyclin D1 transcrip-
tion and CDK2 activity by Notch(ic): implication for cell cycle disruption in
transformation by Notch(ic). Mol. Cell. Biol. 21:5925–5934.
48. Schagger, H., and G. von Jagow. 1987. Tricine-sodium dodecyl sulfate-
polyacrylamide gel electrophoresis for the separation of proteins in the range
from 1 to 100 kDa. Anal. Biochem. 166:368–379.
49. Schroeter, E. H., J. A. Kisslinger, and R. Kopan. 1998. Notch-1 signalling
requires ligand-induced proteolytic release of intracellular domain. Nature
50. Seiffert, D., J. D. Bradley, C. M. Rominger, D. H. Rominger, F. Yang, J. E.
Meredith, Jr., Q. Wang, A. H. Roach, L. A. Thompson, S. M. Spitz, J. N.
Higaki, S. R. Prakash, A. P. Combs, R. A. Copeland, S. P. Arneric, P. R.
Hartig, D. W. Robertson, B. Cordell, A. M. Stern, R. E. Olson, and R.
Zaczek. 2000. Presenilin-1 and -2 are molecular targets for gamma-secretase
inhibitors. J. Biol. Chem. 275:34086–34091.
51. Shawber, C., D. Nofziger, J. J. Hsieh, C. Lindsell, O. Bogler, D. Hayward,
and G. Weinmaster. 1996. Notch signaling inhibits muscle cell differentiation
through a CBF1-independent pathway. Development 122:3765–3773.
52. Struhl, G., and A. Adachi. 1998. Nuclear access and action of notch in vivo.
53. Struhl, G., and I. Greenwald. 2001. Presenilin-mediated transmembrane
cleavage is required for Notch signal transduction in Drosophila. Proc. Natl.
Acad. Sci. USA 98:229–234.
54. Tani, S., H. Kurooka, T. Aoki, N. Hashimoto, and T. Honjo. 2001. The N-
and C-terminal regions of RBP-J interact with the ankyrin repeats of Notch1
RAMIC to activate transcription. Nucleic Acids Res. 29:1373–1380.
55. Taniguchi, Y., H. Karlstrom, J. Lundkvist, T. Mizutani, A. Otaka, M.
Vestling, A. Bernstein, D. Donoviel, U. Lendahl, and T. Honjo. 2002. Notch
receptor cleavage depends on but is not directly executed by presenilins.
Proc. Natl. Acad. Sci. USA 99:4014–4019.
56. Wang, S., S. Younger-Shepherd, L. Y. Jan, and Y. N. Jan. 1997. Only a subset
of the binary cell fate decisions mediated by Numb/Notch signaling in Dro-
sophila sensory organ lineage requires Suppressor of Hairless. Development
57. Weihofen, A., K. Binns, M. K. Lemberg, K. Ashman, and B. Martoglio. 2002.
Identification of signal peptide peptidase, a presenilin-type aspartic protease.
58. Wolfe, M. S. 2001. gamma-Secretase inhibitors as molecular probes of pre-
senilin function. J. Mol. Neurosci. 17:199–204.
59. Wolfe, M. S., W. Xia, B. L. Ostaszewski, T. S. Diehl, W. T. Kimberly, and
D. J. Selkoe. 1999. Two transmembrane aspartates in presenilin-1 required
for presenilin endoproteolysis and gamma-secretase activity. Nature 398:
60. Wu, L., J. C. Aster, S. C. Blacklow, R. Lake, S. Artavanis-Tsakonas, and J. D.
Griffin. 2000. MAML1, a human homologue of drosophila mastermind, is a
transcriptional coactivator for NOTCH receptors. Nat. Genet. 26:484–489.
61. Yan, X. Q., U. Sarmiento, Y. Sun, G. Huang, J. Guo, T. Juan, G. Van, M. Y.
Qi, S. Scully, G. Senaldi, and F. A. Fletcher. 2001. A novel Notch ligand,
Dll4, induces T-cell leukemia/lymphoma when overexpressed in mice by
retroviral-mediated gene transfer. Blood 98:3793–3799.
664WENG ET AL.MOL. CELL. BIOL.