toshifumi morimura , ryo goitsuka , yong zhang , … · toshifumi morimura1, ryo goitsuka1, 2, yong...

1

Cell-cycle arrest and apoptosis induced by Notch1 in B cells

Toshifumi Morimura1, Ryo Goitsuka1, 2, Yong Zhang3, Izumu Saito4,

Michael Reth3, and Daisuke Kitamura1, 5

1Division of Molecular Biology, Research Institute for Biological Sciences, Science

University of Tokyo, 2669 Yamazaki, Noda City, Chiba, 278-0022, Japan

2PRESTO, JST (Japan Science and Technology Corporation), 2669 Yamazaki, Noda City,

Chiba, 278-0022, Japan

3Department for Molecular Immunology, Biology III, University of Freiburg,

and Max Planck Institute for Immunobiology, Stubeweg 51, 79108 Freiburg, Germany

4Laboratory of Molecular Genetics, The Institute of Medical Science, The University of

Tokyo, 4-6-1, Shiroganedai, Minato-ku, Tokyo, 108-8639, Japan

5Corresponding author

Division of Molecular Biology, Research Institute for Biological Sciences, Science University

of Tokyo, 2669 Yamazaki, Noda City, Chiba, 278-0022, Japan

Fax: +471-24-1561

Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on August 30, 2000 as Manuscript M006415200 by guest on July 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

2

Tel: +471-23-9849

E-mail: [email protected]

by guest on July 29, 2018http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

3

Running Title: Notch1 inhibition of B cell proliferation


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

4

Summary

Notch receptors play various roles for cell fate decisions in developing organs, although their

functions at the cell level are little understood. Recently, we found that Notch1 and its ligand

are each expressed in juxtaposed cell-compartments in the follicles of the bursa of Fabricius,

the central organ for chicken B cell development. To examine the function of Notch1 in B

cells, a constitutively active form of chicken Notch1 was expressed in a chicken B cell line,

DT40, by a Cre/loxP-mediated inducible expression system. Remarkably, the active Notch1

caused growth suppression of the cells, accompanied by a cell-cycle inhibition at the G1 phase

and apoptosis. The expression of Hairy1, a gene product up-regulated by the Notch1 signaling,

also induced the apoptosis, but no cell-cycle inhibition. Thus, Notch1 signaling induces

apoptosis of the B cells through Hairy1, and the G1 cell-cycle arrest through other pathways.

This novel function of Notch1 may account for the recent observations indicating the selective

inhibition of early B cell development in mice by Notch1.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

5

Introduction

Notch proteins are a transmembrane receptor family that is structurally and functionally

conserved from worms to humans. Notch was first identified in Drosophila as a gene involved

in neuronal cell fate decision, but this family of receptors is now known to regulate the fate

decisions of developing cells in various tissues during embryogenesis as well as in postnatal

stages (reviewed in 1). Upon binding to its ligand, Notch protein is proteolytically processed

within the transmembrane domain, and its intracellular domain (Notch-IC) is released (2-4).

Notch-IC translocates to the nucleus and acts as a transcriptional activator in cooperation with

a DNA-binding protein, like C promoter binding factor-1 (CBF-1, also known as RBP-Jk ) in

vertebrates (5-8), Suppressor of Hairless in Drosophila (9), or Lag-1 in Caenorhabditis

elegans (10), together termed CSL proteins. Thus, Notch-IC has been used as a ligand-

independent constitutively active form to analyze Notch function in vivo and in vitro (11-14).

The Notch-IC-CSL complex up-regulates the transcription of Enhancer of Split gene encoding

basic helix-loop-helix (bHLH) transcription factors in Drosophila and its mammalian

homologues, Hairy Enhancer of Split (Hes)-1 (15) and Hes-5 (16), through binding to their

promoters. While Hes family proteins are known to be important components of Notch

signaling, Hes-independent pathways are also known to exist in several systems (17-19).

Notch receptors and their ligands are essential for embryogenesis in mice (20-24). In

the hematopoietic system, Notch1 and Notch2 are expressed in CD34+ hematopoietic

progenitor cells (25, 26) and a ligand of Notch is found in a subset of bone marrow cells (27).

Ligand-mediated activation of Notch1 was shown to inhibit G-CSF-induced differentiation of

the myeloid progenitor cell line, 32D (27). In the lymphoid system, Notch1 is strongly

expressed in CD4- CD8- immature thymocytes and its ligands (Jagged1/2) are expressed in

thymic stromal cells (28, 29). By transgenic approach, it has been found that Notch signaling

influences the commitment of CD4/CD8 and αβ/γδ T cell lineages in the thymus (27, 30, 31).


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

6

Recently, it has been reported that the induced deletion of Notch1 gene in adult mice resulted

in the impairment of early T cell development and ectopic development of B cells in the

thymus (32). Conversely, retroviral expression of constitutively active Notch1 in bone marrow

progenitors resulted in a block of early B cell development and the ectopic development of

immature T cells in the bone marrow (33). Thus, Notch1 signaling appears to promote T cell

development as well as to inhibit B cell development of common lymphoid progenitors

primarily destined to develop into B cells.

In mice and humans, B cells are continuously generated in the bone marrow

throughout life. In chickens, however, the B cell generation is restricted to a relatively short

period of life in a lymphoid organ called the bursa of Fabricius (reviewed in 34). The bursa is

composed of about 104 follicles, each of which is colonized by a few B cell precursors

expressing surface immunoglobulin (Ig) M at 8-14 embryonic day (35-38). The precursors

proliferate enormously and their numbers finally reach to 2-5x105/ follicle. During this

process, extensive diversification of Ig is accomplished by Ig gene conversion (39, 40), and

most of the developing B cells die by apoptosis prior to emigration into the periphery (34, 41,

42). The bursa disappears within several months after hatching, and the peripheral B cell pool

is maintained by self-replenishing after the involution of the bursa.

To understand how B cell development and homeostasis are regulated in the chicken

B cell system, we identify molecules specifically expressed in the bursa and study their

functions. Recently, we have found that Notch1 is expressed in the most outer layer of each

follicle in the bursa. The Notch1-expressing cells were surrounded by the cells expressing the

Notch1 ligand, Serrate2, suggesting a possible role for the Notch signaling in B cell

development in the bursa (Goitsuka et al., in preparation). To understand the meaning of their

specific expression pattern and a possible interaction of Notch1 and Serrate2 in the bursa, we

analyzed a function of Notch1 in a bursa-derived B cell line, DT40, by expressing a


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

7

constitutively active form of Notch1 through a new inducible expression system. With this

approach, we have revealed a direct effect of Notch1 signaling in cells, namely, the induction

of cell-cycle arrest at the G1 phase accompanied by apoptosis of the B cell line. In addition,

we demonstrated that Hairy1, a downstream target of Notch signaling, also induces apoptosis,

but not the G1 cell-cycle arrest, of the same cells.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8

Experimental Procedure

Construction of expression vectors. Chicken Notch1ICS gene (corresponding to amino

acids 1748-2142 of rat Notch1) was amplified by PCR with primers, 5'-

CTCAAGCTTGGCGCAAGCGGCGCAGGGAGCATGGCCAGC-3' and

5'-CGGAATTCTAGACGGCCGGCTTCAGGTTGCCGATGTAACTG-3', using a partial

chicken Notch1 cDNA (Goitsuka et al., in preparation) as a template (Fig. 1A). The PCR

product (1.2 kb) was digested with HindIII and EcoRI, cloned into pBlueScript SK+ and

verified for its nucleotide sequence. The HindIII-EcoRI fragment of the Notch1ICS gene was

inserted into a multiple cloning site of pEGFP-C1 (Clontech, Palo Alto, CA) in frame with

GFP sequence to make pEGFP-Notch1ICS or of pAT7neo which contains the chicken β-actin

promoter and double T7 tag in place of the cytomegalovirus promoter/enhancer and GFP

sequences of pEGFP-C1, to make pAT7-Notch1ICS. A cDNA encoding a C-terminal portion

of Notch1 was amplified by PCR with primers based on the reported sequence (43),

5’-GCCCTCTCGGGGCCCCCACGCTGTCCCCCCCGC-3’and

5’-CCCGAATTCACTTGAAGGCCTCGGGGATGTGTCCCAT-3’, using an oligo-dT

primed single strand cDNA library of the bursa. The PCR product (1.3kbp) was cloned into

pGEM-T-Easy (Promega, Madison, WI) and verified for its sequence, from which an ApaI-

EcoRI fragment was excised and inserted into pEGFP-Notch1ICS to make pEGFP-full-length

intracellular region of Notch1 (Notch1ICF). To make pCALNL5-GFP-Notch1ICS, a two kb

Eco47III -EcoRI fragment from the pEGFP-Notch1ICS was inserted into SwaI-EcoRI sites of

pCALNL5, in which a multiple cloning site including SwaI-EcoRI-SacI-KpnI-SmaI sites had

been created at a SwaI site of pCALNLw (44). Similarly, an Eco47III-EcoRI fragment

(3.3kbp) from pEGFP-Notch1ICF was inserted into pCALNL5 to make pCALNL5-GFP-


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

9

Notch1ICF. Chicken Hairy1 cDNA was amplified by PCR with primers based on the reported

sequence (45), 5’-CCGAATTCTATGCCCGCCGACACGGGCATGGAAAAACCCA-3’,

and 5’-CCGGATCCCTACCAGGGGCGCCAGACGGCCTCCCTGCG-3’, using the bursa-

derived cDNA library. The PCR product (0.8kbp) was cloned into pGEM-T-Easy (Promega)

and verified for its sequence, from which an EcoRI-BamHI fragment was excised and inserted

into pEGFP-C1 to make pEGFP- Hairy1. A SacI-BamHI fragment from the pEGFP- Hairy1

was inserted into pBluescript SK+ (pBS- Hairy1). Then, a SacI-KpnI fragment from the pBS-

Hairy1 including Hairy1 cDNA was inserted in pCALNL5-GFP-Notch1ICS in place of

Notch1ICS to make a pCALNL5-GFP- Hairy1. pCAG-Puro-MerCreMer, in which a 3.0 kb

HindIII fragment from pAN-MerCreMer (46) was inserted into an XhoI site of pCAG-Puro,

was provided by Dr. Michinori Kohara. pCAG-Puro was generated by ligating two ScaI-

BamHI fragments from pCAGGS (47) and pBabe-puro (48), containing CAG

enhancer/promoter/poly-A site and a puromycin-resistance gene, respectively (Kohara et al.,

unpublished).

Construction of luciferase reporter vectors. The promoter region of chicken Hairy1 gene

was isolated by screening of λ Fix II genomic library from liver (Stratagene, La Jolla, CA),

and a 0.7 kb fragment containing a CSL-binding motif (GTGGGAA) was subcloned into

pBasic2 vector (Toyo Ink, Tokyo, Japan) at upstream of luciferase gene, to make pBasic2-

Hairy1wt. The chicken Hairy1 genomic DNA sequence has been deposited in

GenBank/EMBL/DDBJ nucleotide database (Accession number AB045236). The same 0.7 kb

fragment but with a deletion of the CSL-binding motif (110bp) was also inserted into the

pBasic2 to make pBasic2-Hairy1mt as a negative control.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

10

Cell lines and transfection. For the analysis of subcellular localization of Notch1-IC, 3µg of

either pEGFP-C1 or pEGFP-Notch1ICS was transfected into NIH3T3 cells (1x105) using

trans IT-LT1 (Mirus, Madison, WI). On the next day, the localization of their gene products

was visually inspected by fluorescence microscopy. NIH3T3 cells were maintained in

Dulbecco’s modified Eagle’s medium (Sigma, Irvine, UK) containing 10 % heat-inactivated

fetal calf serum (FCS; GIBCO BRL, Grand Island, NY) and antibiotics (50U/ml of penicillin

and 50µg/ml of streptomycin, GIBCO BRL). The bursa-derived B lymphoblastoid cell lines,

DT40, and its subline CL18 were cultured in RPMI1640 medium (Sigma) containing 10%

heat-inactivated FCS (GIBCO BRL), 5x10-5M 2-mercaptoethanol (GIBCO BRL) and

antibiotics (GIBCO BRL) as above, at 40˚C. For transfection, cells were washed with the

culture medium, and adjusted to 2x107/ml. Cells (1x107) were transferred to a cuvette and

pulsed at 975µF and 250V in the presence of 30 µg of pAT7neo, pAT7-Notch1ICS, or pCAG-

Puro-MerCreMer. The transfected cells were selected with 1mg/ml of G418 (Wako, Osaka,

Japan) or 1µg/ml of puromycin (Sigma). Drug-resistant clones were subjected to Western

blotting using anti-T7 mouse monoclonal antibody (Novagen, Madison, WI) or anti-Cre rabbit

polyclonal antibody (Novagen). MerCreMer-expressing DT40 cells were further transfected

with either pCALNL5-GFP, pCALNL5-GFP-Notch1ICS, pCALNL5-GFP-Hairy1 or

pCALNL5-GFP-Notch1ICF and selected with 1 mg/ml G418 as above. Drug-resistant clones

were treated for 12 hours with 10 nM of 4-hydroxitamoxifen (OH-TAM; Sigma), an estradiol

analog, and the expression of GFP or GFP-fusion proteins was addressed by flow cytometry

using FACSort (Becton Dickinson, Mountain View, CA) as well as by Western blotting with

anti-GFP mouse monoclonal antibody (Clontech). The resultant clones with negligible

background and the highest induction of the expression of GFP or GFP-fusion proteins were

used for the study.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

11

Luciferase assay. pEGFP-C1, pEGFP-Notch1ICS, pAT7neo or pAT7-Notch1ICS (15µg per

each) were transfected into DT40 cells (5X106) with either pBasic2-Hairy1wt or pBasic2-

Hairy1mt (5 µg per each) and pactβgal (a gift of T. Yagi, 0.5 µg) and, 48 hours later, the cells

were harvested and measured for luciferase and β-galactosidase activities using Luciferase

assay system (Promega) and Galacto-Light (Tropix, Inc., Bedford, MT), respectively, as

described previously (49).

Analysis of cell growth and cell cycle. The stable transfectants of DT40 cells (5x105/ml)

were seeded into 6 well plate (4ml/well) and treated for 12 hours with 10nM OH-TAM to

induce the expression of GFP or GFP-fusion proteins. Then, the number of live cells were

counted using trypan blue and adjusted to 5x105/ml every 24 hours. At the same time, the cells

were also stained with propidium iodide (PI, Sigma) and their DNA contents were analyzed

by FACSort as described previously (50).

Northern blot hybridization. Transcripts of Hairy1 and MerCreMer genes were analyzed by

Northern blot hybridization using the following cDNA probes: a HindIII-BamHI fragment

(0.8kbp) from pBS-Hairy1, and a BamHI-ClaI fragment (0.45kbp) from pCAG-Puro-

MerCreMer. These fragments were labeled with 32P-dCTP by Prime IT II (Stratagene)

according to the supplier's instruction. Total cellular RNAs (5µg per each) extracted with

Trizol (GIBCO BRL) were separated on a 1% agarose gel containing 1% formamide in MOPS

buffer, and transferred to a nylon membrane (Biodyne: Pall, East Hills, NY). Hybridization

was performed as described previously (51).


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

12

Western blot analysis. Protein samples were prepared by boiling cells in SDS sample

buffer. The samples (2.5x105cells/lane) were separated on 7% SDS-PAGE and then

transferred to nitrocellulose membrane. The membrane was probed with anti-GFP mouse

monoclonal antibody (Clontech) or anti-Cre rabbit polyclonal antibody (Novagen). An

enhanced chemiluminescence kit (Amersham Pharmacia, Piscataway, NJ) was used to detect

horseradish peroxidase-labeled secondary antibodies (Zymed, Sun Francisco, CA).


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

13

Results

Construction of a constitutively active form of chicken Notch1. To examine the effect of

Notch1 signaling in chicken B cells, we constructed vectors expressing an intracellular portion

of chicken Notch1 (Notch1ICS, Fig. 1A), which corresponds to a constitutively active form of

mouse Notch1 reported previously (Robey et al., 1996). We then tested whether this chicken

Notch1ICS is indeed active. First, the Notch1ICS was transiently expressed in NIH3T3 cells

as a fusion protein with green fluorescent protein (GFP) and its subcellular localization was

determined. As shown in Fig. 1B, the GFP-Notch1ICS protein was found exclusively in the

nuclei, whereas the control GFP was distributed diffusely in the cell. The same result was

obtained with the chicken B cell line, DT40 (data not shown). We next examined the ability of

Notch1ICS in DT40 cells to activate the transcription from the promoter of chicken Hairy1

gene which is one of Hes family genes and has a CSL protein binding motif in its promoter

region (GenBank/EMBL/DDBJ, Accession Number AB045236). In comparison to the GFP

control, the GFP-Notch1ICS strongly activated the chicken Hairy1 gene promoter, but not the

mutant promoter with a deletion of the CSL protein binding site (Fig. 1C). Notch1ICS with a

T7-tag at the N-terminus similarly activated the Hairy1 promoter. These results indicate that

the Notch1ICS is constitutively active in chicken B cells.

Growth-inhibitory effect of Notch1ICS in bursa-derived B-lymphoblastoid cell lines. To

reveal a function of Notch1 in developing B cells in the bursa, we utilized a bursa-derived B-

lymphoblastoid cell line, DT40, as a model system. DT40 cells maintain the characteristics of

the bursal B cells: namely, expression of specific surface markers such as ChB1 (52), and

more significantly, continuous somatic gene conversion at Ig loci (53). DT40 cells express

both Notch1 and Serrate2 weakly, but not Hairy1, suggesting that self ligand-receptor

interaction, if any, is not enough to evoke intracellular signaling (Goitsuka et al., in


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

14

preparation; see Fig. 6). To address the function of Notch1, we transfected DT40, or its

surface IgM-negative variant, CL18, with T7-tagged Notch1ICS expression vector. Despite

repeated transfection experiments, however, we could not establish any stable transfectants

from DT40, and only one from CL18 expressing Notch1ICS, which showed very slow

proliferation rate (data not shown). Cell-cycle analysis of the Notch1ICS-transfectant showed

that it contained high proportion of apoptotic cells, compared to the parental or mock-

transfected CL18 cells (Fig. 2). Similar effect of the Notch1ICS was observed in a few

transfectants obtained from 249L4 cells, another bursa-derived B cell line expressing Notch1

(data not shown). Thus, Notch1ICS expression appears to be lethal for the B cell lines tested

above, and occasionally arising transfectants suffer a severe growth disadvantage with

continuous apoptosis in a fraction of cells.

Induced expression of Notch1ICS causes G1 cell-cycle inhibition and apoptosis in DT40 cell

line. To avoid the cell lethality by the constitutive expression of Notch1ICS and to analyze

the direct effects of Notch1ICS on the proliferation and/or survival of the cells, we applied a

Cre/loxP-mediated inducible system (54) for the expression of Notch1ICS. An expression

vector encoding the chimeric Cre-recombinase, MerCreMer, with a ligand binding domain of

the mouse estrogen receptor at both ends was first introduced into DT40 cells. A stable

transfectant strongly expressing the MerCreMer protein was selected by Western blotting,

then transfected with the expression vectors in which GFP or GFP-Notch1ICS fusion genes

were separated from the promoter by loxP-flanked neomycin-resistance gene (Fig. 3A). In the

resultant transfectants (GFP or GFP-Notch1ICS clones), loxP-mediated recombination and the

following expression of GFP or GFP-Notch1ICS genes can be induced by treatment with OH-

TAM. Flow cytometric analysis showed that more than 97 % of the transfectants expressed

either GFP or GFP-Notch1ICS proteins upon treatment with 10 nM of OH-TAM for 12 hours


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

15

(Fig. 3B). Two of the GFP-Notch1ICS clones (N#1-1, N#3-1) and a control GFP clone (G#1-

13), each showing negligible fluorescence before OH-TAM treatment, were used for further

analyses.

Since OH-TAM itself inhibited the growth of DT40 cells slightly (data not shown),

transfectants were treated with OH-TAM for the first 12 hours to induce the expression of

transgenes, then washed and further cultured in normal medium. The GFP-Notch1ICS clones

proliferate constantly in the absence of Notch1ICS expression, but the proliferation was

gradually inhibited by the induction of Notch1ICS, which first became evident 3 days after the

induction (Fig. 4B and C). The control GFP clone proliferates at a constant rate irrespective

of the induction of GFP (Fig. 4A).

To characterize the growth regulation by Notch1ICS, cell-cycle profile was analyzed

by flow cytometry every 24 hours of the culture after the induction of Notch1ICS expression

(Fig. 5A). The proportion of apoptotic cells increased gradually and reached over 25% by day

4 after the expression of Notch1ICS (a). The proportion of the cells in the S phase started to

decrease at day 2 and became about half of the starting population by day 4 (c), while that in

the G2/M phase slightly decreased (d). The proportion of the cells in the G0/G1 phase was

roughly unchanged during the experimental period (b), indicating a relative increase of this

population among live cells. Control cells expressing GFP did not grossly alter their cell cycle

profile in these culture periods. Representative cell-cycle profiles on day 4 is shown in Fig.

5B. The ratio of the proportions of the cells in the G0/G1 versus S phases significantly

increased by expression of the Notch1ICS, but not by expression of the GFP. These results

indicate that Notch1 signaling inhibits cell-cycle progression at the G1 phase and induces

apoptosis of DT40 cells, although the kinetics of these changes appear to be rather slow

compared to that of apoptosis of the same cells induced by surface IgM cross-linking (55).


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

16

Induced expression of Hairy1 causes apoptosis in DT40 cell line. In mice, Hes1 gene is the

best-characterized target of Notch signaling and its transcription is up-regulated by Notch-IC-

CSL complex bound to its promoter region. We have also demonstrated that Hairy1gene

promoter was transactivated by Notch1-ICS in a CSL-binding site dependent manner (Fig.

1C). Accordingly, the level of endogenous Hairy1 transcript was up-regulated by the

induction of Notch1ICS expression in the DT40 cell transfectants (Fig. 6A). Therefore, it is

possible to speculate that the Hairy1 protein mediates the Notch1 signaling that induces cell-

cycle inhibition and/or apoptosis of DT40 cells. To test this possibility, we next generated the

DT40 transfectants in which the expression of GFP-Hairy1 fusion protein can be induced by

the same vector system as described above (Fig. 3A). The expression of the GFP-Hairy1

fusion mRNA was induced by OH-TAM treatment in the transfectants to almost the same

level as the endogenous Hairy1 mRNA induced by Notch1ICS (Fig. 6A). The induction of the

GFP-Hairy1 expression resulted in growth suppression as evident already on day 1, and the

number of live cells did not increase after day 2 (Fig. 6B). Cell-cycle analysis revealed that the

proportion of the apoptotic cells obviously increased 2 days after the Hairy1 induction and

reached to 45% by day 4 (Fig. 6C). In sharp contrast to the case of Notch1ICS-induction, the

proportion of the cells in the G0/G1 phase started to decrease as early as day1 and continued

to decrease until day 4, whereas that in the S/G2/M phases did not change markedly by day 2

and later decreased in proportion to the increase of apoptotic cells. Thus, the induction of

Hairy1-expression caused earlier and stronger apoptosis compared to that induced by

Notch1ICS, although it did not cause G1 cell-cycle inhibition.

We also tried to establish stable transformants by repeated transfections of

conventional constitutive expression vectors encoding T7-tagged Hairy1. However, we failed

to obtain a single clone from DT40 cells, and obtained several clones from 249L4 cells all of


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

17

that showed a marked apoptosis in culture and gradual decrease of Hairy1 expression and/or

outgrowth of Hairy1-negative variants (data not shown).

C-terminal part of Notch1 are necessary for full activity of Notch1 intracellular domain to

induce G1 cell-cycle arrest and apoptosis. Above results are consistent with a scenario in

which the Notch1ICS complexed with CSL protein up-regulates the expression of Hairy1

gene, and then the Hairy1 protein regulates genes to induce apoptosis, whereas other

mediator(s) than Hairy1 being activated by Notch1ICS induces cell-cycle arrest at the G1

phase. However, the Notch1ICS-induced apoptosis and G1-arrest appeared to be rather

modest in terms of the timing of onset and the extent (Fig. 4 and 5). We thought this might be

due to the lack of the C-terminal amino acids of Notch1 in the Notch1ICS used above.

Therefore, we next constructed an inducible expression vector coding for a full-length

intracellular portion of Notch1 (Notch1ICF) as a GFP-fusion protein, and induced its

expression in DT40 cells in the same system as above (Fig. 3A). The GFP-Notch1ICF

localized in the nucleus of the cells and induced the Hairly1 promoter activity as well as the

expression of the endogenous Hairy1 gene to the similar level to that GFP-Notch1ICS did

(data not shown, Fig. 6A). The expression level of GFP-Notch1ICF protein was less than that

of GFP-Notch1ICS protein after induction (Fig. 7A). Nevertheless, the induction of the

Notch1ICF resulted in a strong cell-cycle arrest at G1 phase (Fig. 7B). Proportion of cells in

the G1 phase started to increase and that in the S/G2/M to decrease already in the induction

period (see day 0), and most of live cells were arrested at the G1 phase as early as on day 1.

The Notch1ICF expression also strongly induced apoptosis as evident already on day 2 and

the proportion of the apoptotic cells increased thereafter. The G1 cell-cycle arrest and

apoptosis induced by the Notch1ICF were much stronger and more rapid than those by the

Notch1ICS shown above (Fig. 5). These results indicated that the full length of the Notch1


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

18

intracellular domain is required for its full activity to induce apoptosis and G1 cell-cycle

arrest, not affecting the level of transactivation of Hairy1 gene.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

19

Discussion

To understand the biological function of Notch1 in developing B cells in the bursa of

Fabricius of chicken, we examined the effects of an active form of chicken Notch1 in a bursa-

derived B lymphoblastoid cell line, DT40 cells, using a new inducible expression system. Our

results indicate that the Notch1 signal induces both apoptosis and G1 cell-cycle arrest in the B

cells, whereas Hairy1 only mediates apoptosis. In addition, we have found that the C-terminal

region of Notch1, including PEST domain, is necessary for a full activity.

A human homologue of Notch, TAN1, was first identified as a proto-oncogene whose

intracellular region was translocated into the T cell antigen receptor (TCR) β locus in T

lymphoblastic leukemia/lymphomas (56). The ability of TAN1-IC to induce T-cell leukemia

was confirmed in mice by its retroviral introduction into mouse bone marrow cells (57). In

addition, Notch1-IC was shown to rescue thymoma cells and T-cell hybridomas from

glucocorticoid- and TCR-mediated apoptosis, respectively (58, 59). These observations

suggest that Notch1-IC is able to promote the proliferation and survival of cells of the T-cell

lineage, in sharp contrast to its activities in B cells as described in this paper. Such an opposite

function of Notch1 signaling between T and B cell-lineages may account for the recent

findings that the Notch1 signaling is necessary for the T cell development, whereas it inhibits

B cell development in mice (32, 33). Thus, shortly after the T/B-lineage commitment of

lymphoid progenitors, Notch1 signaling may promote proliferation and differentiation of T

cell progenitors, whereas inhibit those of B cell progenitors.

Recently, it was shown that Hes1 is necessary for the self-renewal of multipotent

progenitor cells in mouse brain (60) and for expansion of early T cell precursors in mouse

thymus (61), indicating that Hes1 promotes proliferation of these cells. Our results, however,

demonstrate that Hairy1, whose expression was up-regulated by the Notch1-IC, induced


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

20

apoptosis of the B cell lines. Thus, these two proteins may have different effects in controlling

apoptosis in spite of their sequence homology. Alternatively, the function of Hes family

proteins may be cell-type specific.

Over-expression of Hairy1 in DT40 cells resulted in earlier and severer apoptosis

compared to that caused by Notch1ICS, although the expression level of the GFP-Hairy1

gene in the former was equivalent to that of the endogenous Hairy1 gene induced by the latter.

This suggests that Notch1ICS may also signals to attenuate the function of Hairy1,

presumably through translational or post-translational modification of Hairy1, or through

affecting the downstream of Hairy1 in the pathway leading to apoptosis. On the other hand,

Hairy1 did not cause G1 cell-cycle arrest in contrast to Notch1ICS or Notch1ICF, indicating

that the Notch1-IC-induced G1 cell-cycle arrest is mediated by Hairy1-independent

mechanism(s). Deltex is another Notch-binding protein whose structure and function are

conserved from flies to humans. It functions as a positive regulator of Notch signaling (62,

63). The Deltex expression is up-regulated by Notch1-IC (58) and Deltex inhibits the

transcriptional activity of E47, a bHLH protein that is necessary for early B cell development

(18, 63). In B cells, E47 homodimers activate transcription of several genes which are critical

for B cell development, such as IgH, λ5, VpreB, Rag1 (64-66), as well as B cell-specific

activator protein (BSAP)/Pax5 (66, 67). BSAP/Pax5 is a transcription factor also necessary for

early B cell development (68, 69). It was observed that the BSAP/Pax5 gene was strongly

expressed in B cell lymphomas through its translocation into Ig heavy chain locus (70, 71). In

addition, antisense oligonucleotide-mediated suppression of BSAP activity caused growth

inhibition of B cells, but not of T lymphoma or plasma cell lines (72). These observations

indicate that BSAP/Pax5 promotes proliferation of B cells. Taken together, it seems possible

to imagine a scenario in which Notch1 signaling inhibits E47 activity through Deltex, and thus


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

21

down-regulates BSAP/Pax5 expression, resulting in growth inhibition of B cells. The

experiments to test this possibility are in progress with our DT40 cell system.

We demonstrated here that the C-terminal region of Notch1 including the PEST

domain strongly enhanced the induction of G1 cell-cycle arrest and apoptosis by Notch1

signaling in DT40 cells. It was previously shown that C-terminal region of mouse Notch1

protein acts as positive regulatory domain for CSL-dependent transcriptional activation and

works as transactivation domain by itself (73). Although Hes1gene expression is known to be

activated by CSL, we have observed no significant enhancement of Notch1-IC-induced

endogenous Hairy1 expression as well as Hairy1 gene promoter activation by the addition of

the C-terminal region (Fig.6A, data not shown). Therefore, a role of the C-terminal domain in

CSL-mediated transcriptional activation may differ among the target genes. To support this,

Schroeder and Just reported that presence/absence of the C-terminal domain of mouse Notch1

did not affect the CSL-mediated enhancement of G-CSF-induced differentiation of a myeloid

progenitor cell line (74). In our system, it seems that the C-terminal region of chicken Notch1

positively regulated transactivation of certain genes responsible for G1 cell-cycle arrest and

apoptosis.

A few B-cell precursors undergo extensive proliferation and Ig gene conversion in the

bursa to enlarge their diversity. It was estimated, however, that the vast majority of the newly

generated B cells die in situ, and that only 5% of the cells generated daily emigrate to the

periphery (41, 42). In the periphery, mature B cells are resting until they encounter the antigen.

As mentioned above, B cells located in the most outer layer of each follicle in the bursa

express Notch1, and are surrounded by the cells expressing the Notch1 ligand (Goitsuka et al.,

in preparation). Therefore, it is possible to speculate that the B cells are arrested in their

proliferation by Notch1 signaling and die by apoptosis unless some survival signal is


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

22

provided. In this way, Notch1 might contribute to select functional B cells and to promote

maturation of B cells before emigration into the periphery.

Acknowledgements

We thank Drs. J.-I. Miyazaki for CAG promoter, M. Kohara for pCAG-Puro-MerCreMer, T.

Yagi for pactβgal, K. Ohashi for RNA sample of the bursa, and all members of Kitamura lab

for materials. This work was supported by Special Coordination Funds for Promoting Science

and Technology from Science and Technology Agency in Japan and Research Fellowships of

the Japan Society for the Promotion of Science for Young Scientists.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

23

References

1. Artavanis-Tsakonas, S., Matsuno, K, and Fortini, M. E. (1995) Science 268, 225-232

2. Kidd, S., Lieber, T., and Young, M. W. (1998) Genes Dev. 23, 3728-3740

3. Schroeter, E.H., Kisslinger, J. A., and Kopan, R. (1998) Nature 393, 382-386

4. Struhl, G., and Adachi, A. (1998) Cell 93, 649-660

5. Furukawa, T., Maruyama, S., Kawaichi, M., and Honjo, T. (1992) Cell 69, 1191-1197

6. Honjo,T. (1996) Genes Cells 1, 1-9

7. Matsunami, N., Hamaguchi, Y., Yamamoto, Y., Kuze, K., Kangawa, K., Matsuo, H.,

Kawaichi, M., and Honjo, T. (1989) Nature 342, 934-937

8. Schweisguth, F., and Posakony, J. W. (1992) Cell 69, 1199-1212

9. Fortini, M. E., and Artavanis-Tsakonas, S. (1994) Cell 79, 273-282

10. Christensen, S., Kodoyianni, V., Bosenberg, M., Friedman, L., and Kimble, J. (1996)

Development 122, 1373-1383

11. Coffman, C.R., Skoglund, P., Harris, W. A., and Kintner, C. R. (1993) Cell 73, 659-671

12. Lieber, T., Kidd, S., Alcamo, E., Corbin, V., and Young, M. W. (1993) Genes Dev. 10,

1949-1965

13. Rebay, I., Fehon, R. G., and Artavanis-Tsakonas, S. (1993) Cell 74, 319-329

14. Struhl, G., Fitzgerald, K., and Greenwald, I. (1993) Cell 74, 331-345

15. Jarriault, S., Brou, C., Logeat, F., Schroeter, E. H., Kopan, R., and Israel, A. (1995) Nature

377, 355-358

16. Ohtsuka, T., Ishibashi, M., Gradwohl, G., Nakanishi, S., Guillemot, F., and Kageyama, R.

(1999) EMBO J. 18, 2196-2207

17. Nofziger, D., Miyamoto, A. Lyons, K. M., and Weinmaster, G. (1999) Development 126,

1689-1702


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

24

18. Ordentlich, P., Lin, A., Shen, C. B., Blaumueller, C., Matsuno, K., Artavanis-Tsakonas,

S., and Kadesch, T. (1998) Mol.Cell. Biol. 18, 2230-2239

19. Oswald, F., Liptay, S., Adler, G., and Schmid, R. M. (1998) Mol. Cell. Biol. 18, 2077-

2088

20. Conlon, R. A., Reaume, A. G., and Rossant, J. (1995) Development 121, 1533-1545

21. Hamada, Y., Kadokawa, Y., Okabe, M., Ikawa, M., Coleman, J.R., and Tsujimoto, Y.

(1999) Development 126, 3415-3424

22. Jiang, R., Lan, Y., Chapman, H. D., Shawber, C., Norton, C. R., Serreze, D. V.,

Weinmaster, G., and Gridley, T. (1998) Gene Dev. 12, 1046-1057

23. Swiatek, P. J., Lindsell, C. E., del Amo, F. F., Weinmaster, G., and Gridley, T. (1994)

Genes Dev. 8, 707-719

24. Xue, Y., Gao, X., Lindsell, C. E., Norton, C. R., Chang, B., Hicks, C., Gendron-Maguire,

M., Rand, E. B., Weinmaster, G., and Gridley, T. (1999) Hum. Mol. Genet. 8, 723-730

25. Milner, L.A., Kopan, R., Martin, D. I., and Bernstein, D. (1994) Blood 83, 2057-2062

26. Varnum-Finney, B., Purton, L. E., Yu, M., Brashem-Stein, C., Flowers, D., Staats, S.,

Moore, K. A., Le Roux,, I., Mann, R., Gray, G., Artavanis-Tsakonas, S., and Bernstein,

I. D. (1998) Blood 91, 4084-4091

27. Li, L., Milner, L. A., Deng, Y., Iwata, M., Banta, A., Graf, L., Marcovina, S., Friedman,

C., Trask, B. J., Hood, L., and Torok-Storb, B. (1998) Immunity 8, 43-55

28. Hasserjian, R. P., Aster, J. C., Davi, F., Weinberg, D. S., and Sklar, J. (1996) Blood 88,

970-976

29. Felli, M. P., Maroder, M., Mitsiadis, T. A., Campese, A. F., Bellavia, D., Vacca, A.,

Mann, R. S., Frati, L., Lendahl, U., Gulino, A., and Screpanti, I. (1999) Int. Immunol.

11, 1017-1025


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

25

30. Robey, E., Chang, D., Itano, A., Cado, D., Alexander, H., Lans, D., Weinmaster, G., and

Salmon, P. (1996) Cell 87, 483-492

31. Washburn, T., Schweighoffer, E., Gridley, T., Chang, D., Fowlkes, B. J., Cado, D., and

Robey, E. (1997) Cell 88, 833-843

32. Radtke, F., Wilson, A., Stark, G., Bauer, M., van Meerwijk, J., MacDonald, H. R., and

Aguet, M. (1999) Immunity 10, 547-558

33. Pui ,J. C., Allman, D., Xu, L., DeRocco, S., Karnell, F. G., Bakkour, S., Lee, J. Y.,

Kadesch, T., Hardy, R. R., Aster, J. C., and Pear, W. S. (1999) Immunity 11, 299-308

34. Masteller, E. L., Pharr, G. T., Funk, P. E., and Thompson, C. B. (1997) Int. Rev. Immunol.

15, 185-206

35. Houssaint, E., Torano, A., Ivanyi, J. (1983) Eur. J. Immunol . 7, 590-595

36. Pink, J. R., Ratcliffe, M. J., and Vainio, O. (1985) Eur. J. Immunol. 15, 617-620

37 Ratcliffe, M. J., Lassila, O., Pink, J. R., and Vainio, O. (1986) Eur. J. Immunol. 16, 129-

133

38. Weill, J. C., Reynaud, C. A., Lassila, O., and Pink, J. R. (1986) Proc. Natl. Acad. Sci.

USA. 83, 3336-3340

39. Reynaud, C. A., Anquez, V., Grimal, H., and Weill, J. C. (1987) Cell 48, 379-388

40. Reynaud, C. A., Dahan, A., Anquez, V., and Weill, J. C. (1989) Cell 59, 171-183

41. Lassila, O. (1989) Eur. J. Immunol. 19, 955-958

42. Motyka, B., and Reynolds, J. D. (1991) Eur. J. Immunol. 21, 1951-1958

43. Wakamatsu Y., Maynard, T. M., Jones, S. U., and Weston, J. A. (1999) Neuron 23, 71-81

44. Kanegae, Y., Takamori, K., Sato, Y., Lee, G., Nakai, M., and Saito, I. (1996) Gene 181,

207-212

45. Palmeirim, I., Henrique, D., Ish-Horowicz, D., and Pourquie, O. (1997) Cell 91, 639-648


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

26

46. Zhang, Y., Riesterer, C., Ayrall, A. M., Sablitzky, F., Littlewood, T. D., and Reth, M.

(1996) Nucleic. Acids Res. 24, 543-548

47. Sakai, K., Mitani, K., and Miyazaki, J. (1995) Biochem. Biophys. Res. Commun. 217, 393-

401

48. Morgenstern, J. P., and Land, H. (1990) Nucleic. Acids Res. 18, 3587-3596

49. Katsuta, H., Tsuji, S., Niho, Y., Kurosaki, T., and Kitamura, D. (1998) J. Immunol. 160,

1547-1551

50. Fukuda, T., Kitamura, D., Taniuchi, I., Maekawa, Y., Benhamou, L. E., Sarthou, P., and

Watanabe, T. (1995) Proc. Natl. Acad. Sci. USA. 92, 7302-7306

51. Morimura, T., Hattori, M., Ohashi, K., Sugimoto, C., and Onuma, M. (1995) J. Gen.

Virol. 76, 2979-2985

52. Goitsuka, R., Chen, C. H., and Cooper, M. D. (1997) J. Immunol.159, 3126-3132

53. Buerstedde, J. M., Reynaud, C. A., Humphries, E. H., Olson, W., Ewert, D. L., and Weill,

J. C. (1990) EMBO J. 9, 921-927

54. Zhang, Y., Wienands, J., Zurn, C., and Reth, M. (1998) EMBO J. 17: 7304-7310

55. Takata, M., and Kurosaki, T. (1996) J. Exp. Med. 184, 31-40

56. Ellisen, L.W., Bird, J., West, D. C., Soreng, A. L., Reynolds, T. C., Smith, S. D., and

Sklar, J. (1991) Cell 66, 649-661

57. Pear, W. S., Aster, J. C., Scott, M. L., Hasserjian, R. P., Soffer, B., Sklar, J., and

Baltimore, D. (1996) J. Exp. Med. 183, 2283-2291

58. Deftos, M. L., He, Y. W., Ojala, E. W., and Bevan, M. J. (1998) Immunity 9, 777-786

59. Jehn, B. M., Bielke, W., Pear, W. S., and Osborne, B. A. (1999) J. Immunol. 162, 635-638

60. Nakamura, Y., Sakakibara, Si., Miyata, T., Ogawa, M., Shimazaki, T., Weiss, S.,

Kageyama, R., and Okano, H. (2000) J. Neurosci. 20, 283-293


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

27

61. Tomita, K., Hattori, M., Nakamura, E., Nakanishi, S., Minato, N., and Kageyama, R.

(1999) Genes Dev. 13, 1203-1210

62. Matsuno, K., Diederich, R. J., Go, M. J., Blaumueller, C. M., and Artavanis-Tsakonas, S.

(1995) Development 121, 2633-2644

63. Matsuno, K., Eastman, D., Mitsiades, T., Quinn, A. M., Carcanciu, M. L., Ordentlich, P.,

Kadesch, T., and Artavanis-Tsakonas, S. (1998) Nat. Genet. 19, 74-78

64. Choi, J. K., Shen, C. P., Radomska, H. S., Eckhardt, L. A., and Kadesch, T. (1996) EMBO

J. 15, 5014-521

65. Sigvardsson, M., O'Riordan, M., and Grosschedl, R. (1997) Immunity 7, 25-36

66. O'Riordan, M., and Grosschedl, R. (1999) Immunity 11, 21-31

67. Bain, G., Maandag, E. C., Izon, D. J., Amsen, D., Kruisbeek, A. M., Weintraub, B. C.,

Krop, I., Schlissel, M. S., Feeney, A. J., van Roon, M., van der Valk, M., te Riele, H.

P. J., Berns, A., and Murre, C. (1994) Cell 79, 885-892

68. Nutt, S. L., Thevenin, C., and Busslinger, M. (1997) Immunobiol. 198, 227-235

69. Urbanek, P., Wang, Z. Q., Fetka, I., Wagner, E. F., and Busslinger, M. (1994) Cell 79,

901-912

70. Hamada, T., Yonetani, N., Ueda, C., Maesako, Y., Akasaka, H., Akasaka, T., Ohno, H.,

Kawakami, K., Amakawa, R., and Okuma, M. (1998) Br. J. Haematol. 102, 691-700

71. Morrison, A. M., Jager, U., Chott, A., Schebesta, M., Haas, Q. A., and Busslinger, M.

(1998) Blood 92, 3865-3878

72. Wakatsuki, Y., Neurath, M. F., Max, E. E., and Strober, W. (1994) J. Exp. Med. 179,

1099-1108

73. Kurooka, H., Kuroda, K., and Honjo, T. (1998) Nucleic. Acids Res. 26, 5448-5455

74. Schroeder, T., and Just, U. (2000) EMBO J. 19, 2558-2568


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

28

Footnotes

Abbreviations

Notch1-IC: intracellular proportion of Notch

bHLH: basic helix-loop-helix

Hes: hairy enhancer of split

CSL: CBF-1, suppressor of hairless, lag-1

OH-TAM: 4-hydroxitamoxifen

GFP: Green fluorescence protein

Ig: immunoglobulin


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

29

Figure Legends

Figure 1. Construction of a constitutively active form of chicken Notch1. (A) Structure of

full-length chicken Notch1 (top) and Notch1ICS (bottom). EGFR, epidermal growth factor

like -repeats; LNR, Lin12/Notch repeats; TM, transmembrane region; RAM, CSL-binding

site; ANKR, cdc10/ankyrin repeats; NLS, putative nuclear localization signals; PEST, proline-

glutamate-serine-threonine-rich domain. Notch1ICS consists of the intracellular region

containing the first NLS, RAM, and ANKR. (B) Subcellular localization of GFP-Notch1ICS

protein. NIH3T3 cells were transfected with either pEGFP-C1 (a) or pEGFP-Notch1ICS (b)

and their green fluorescence was inspected by fluorescence microscopy after 24 hours. (C)

Transactivation of chicken Hairy1 promoter by Notch1ICS. The luciferase reporter vectors

containing wild-type Hairy1 promoter (pBasic2-Hairy1wt; a) or Hairy1 promoter with a

deletion of the CSL-binding site (pBasic2-Hairy1mt; b) were cotransfected into DT40 cells

with no vectors (lane 1), control vectors (pEGFP-C1, lane 2; pAT7neo, lane 4) or Notch1ICS-

expressing vectors (pEGFP-Notch1ICS, lane 3; pAT7-Notch1ICS, lane 5), and with a

standard vector, pactβgal. Luciferase and β-galactosidase activities in triplicated sample 48

hours after transfection were measured and the luciferase activities were normalized by the β-

galactosidase activities. Each bar represents the mean with SD of the fold induction of the

normalized value over that shown in lane 1.

Figure 2. Apoptotic phenotype of a stable transfectant constitutively expressing Notch1ICS.

Parental CL18 cells (A), mock-transfected (B) and stable Notch1ICS transfectant (C) cells

were permeabilized, stained with PI and analyzed by flow cytometry for their DNA contents.

The numbers indicate the proportions of cells in the subdiploid (apoptotic), G0/G1, S and


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

30

G2/M phases of cell cycle (left to right). Shown is a representative data of three independent

analyses with essentially identical results.

Figure 3. Cre/loxP-mediated inducible expression of GFP and GFP-Notch1ICS. (A) A

scheme of the inducible expression system. In the absence of OH-TAM, the fusion

recombinase MerCreMer is kept inactive by heat shock protein (Hsp) 90 binding. OH-TAM

liberates MerCreMer from Hsp90 and activates it, resulting in the removal of a loxP-flanked

neo-cassette from the inducible expression vector and transcription of GFP-Notch1ICS. (B)

OH-TAM-induced expression of GFP (left) or GFP-Notch1ICS (right) proteins in stable

DT40 cell transfectants. Stable clones carrying the MerCreMer vector and the inducible

expression vector, encoding either GFP or GFP-Notch1ICS, were cultured in the presence of

10 nM of OH-TAM for the indicated periods of time. The GFP-fluorescence intensity of these

clones was measured by FACSort. The percentages of GFP-positive cells (within the gate

shown as a horizontal bar in each histogram) at the indicated time points are denoted.

Figure 4. Growth suppression of DT40 cells by the expression of Notch1ICS. A control GFP

clone (A, G#1-13) and two GFP-Notch1ICS clones (B, N #1-1; C, N#3-1) were cultured with

(closed circle) or without (open circle) 10 nM OH-TAM for 12 hours, then washed (day 0),

and continued to be cultured in the medium without OH-TAM. The density of live cells was

determined and reset to 5x 105/ml in the cultures every 24 hour. Plotted are the means of

calculated cumulative numbers of live cells at indicated time points, derived from three

independent experiments.

Figure 5. Induction of apoptosis and G1 cell-cycle arrest of DT40 cells by the expression of

Notch1ICS. (A) Two GFP-Notch1ICS clones (N#1-1, closed circle; N#3-1, closed triangle)


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

31

and control GFP clone (G#1-13, open circle) were treated with 10 nM OH-TAM as in Fig. 4.

Before the OH-TAM treatment (Pre), 12 hours after the treatment (day 0) and every 24 hours

in the following culture without OH-TAM, cells were stained with PI for their DNA contents

and analyzed by FACSort. Plotted are the means of the percentages of the cells in the

subdiploid (apoptotic) (a), G0/G1 (b), S (c) and G2/M (d) phases, derived from three

independent experiments. (B) A representative result of the above analysis on day 4 is shown

as histograms. The indicated clones were cultured with (right panels) or without (left panels)

OH-TAM. The numbers over the horizontal bars indicate the proportions of cells in the

subdiploid (apoptotic), G0/G1, S and G2/M phases of cell cycle (left to right). G1/S ratio, the

ratio of the cells in the G0/G1 versus S phases.

Figure 6. Induction of apoptosis by the expression of Hairy1. (A) Hairy1 gene expression in

the stable cell lines. Total cellular RNAs were extracted from GFP, GFP-Notch1ICS, GFP-

Notch1ICF and GFP-Hairy1 clones cultured with (+) or without (-) 10 nM OH-TAM for 12

hours, and subjected to Northern blot hybridization with a Hairy1 cDNA probe (upper). The

same filter was reprobed with Cre gene fragment to verify the amount of loaded RNA (lower).

(B, C) Kinetic analysis for cell growth (B) and cell cycle (C) of a stable DT40 cell transfectant

carrying a Hairy1-inducible vector. (B) After induction of GFP-Hairy1 expression by OH-

TAM (day 0), cells were cultured and analyzed as in Fig. 4. (C) Before (-) or after induction of

GFP-Hairy1 by OH-TAM, cells were cultured and analyzed for their DNA contents as in Fig.

5. Shown is a representative result of three independent experiments with one stable clone and

several experiments with other clones, all of which showed similar results.

Figure 7. Induction of apoptosis and G1 cell-cycle arrest of DT40 cells by the induced

expression of Notch1ICF. (A) Comparison of the expression level of Notch1ICF protein with


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

32

Notch1ICS protein. The stable transformants were cultured with OH-TAM, and then lysed in

SDS sample buffer. Protein samples were subjected to Western blotting probed with anti-GFP

antibody (upper). The same filter was reprobed with anti-Cre antibody to verify the amount of

loaded protein (lower). (B) Kinetic analysis for cell cycle of a stable DT40 cell transfectant

carrying a Notch1ICF-inducible vector. The cells were treated with OH-TAM, cultured and

analyzed for their DNA contents as in Fig. 6 (C). Shown is a representative result of three

independent experiments with one stable clone and several experiments with other clones, all

of which showed similar results.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Daisuke KitamuraToshifumi Morimura, Ryo Goitsuka, Yong Zhang, Izumu Saito, Michael Reth and

Cell-cycle arrest and apoptosis induced by Notch1 in B cells

published online August 30, 2000J. Biol. Chem.

10.1074/jbc.M006415200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M006415200

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M006415200v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2000/08/30/jbc.M006415200.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2000/08/30/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2000/08/30/jbc.M006415200.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/

toshifumi morimura , ryo goitsuka , yong zhang , … · toshifumi morimura1, ryo goitsuka1, 2, yong...

Documents