Deregulation of Cdc2 kinase induces caspase-3activation and apoptosis

Ling Gu,a Hongwu Zheng,a Stephen A. Murray,a

Haoqiang Ying,a and Zhi-Xiong Jim Xiaoa,b,*

a Departments of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USAb Departments of Medicine, Boston University School of Medicine, Boston, MA 02118, USA

Received 13 January 2003

Abstract

Progression of the cell cycle and control of apoptosis are tightly linked processes. It has been reported that manifestation of

apoptosis requires cdc2 kinase activity yet the mechanism(s) of which is largely unclear. In an attempt to study the role of human

MDM2 (HDM2) in interphase and mitosis, we employed the Xenopus cell-free system to study HDM2 protein stability. Interest-

ingly, HDM2 is specifically cleaved in Xenopus mitotic extracts but not in the interphase extracts. We demonstrate that HDM2

cleavage is dependent on caspase-3 and that activation of cdc2 kinase results in caspase-3 activation in the Xenopus cell-free system.

Furthermore, expression of cdc2 kinase in mammalian cells leads to activation of caspase-3 and apoptosis. Taken together, these

data indicate that deregulation of cdc2 kinase activity can trigger apoptotic machinery that leads to caspase-3 activation and

apoptosis.

� 2003 Elsevier Science (USA). All rights reserved.

Keywords: Cdc2; Caspase; Apoptosis; Xenopus; HDM2

Apoptosis, or programmed cell death, is a highly

regulated process in elimination of unwanted or dam-

aged cells from multicellular organisms. Deregulation of

this process is implicated in various diseases including

cancer [1–3]. The biochemical hallmarks of apoptosis

include activation of the interleukin-1b-converting en-zyme (ICE) family of proteases [4–6], chromatin con-

densation, and DNA fragmentation [7]. Many differentcell types undergo apoptosis in response to various dif-

ferent extracellular and intracellular stimuli, such as

DNA damage, withdrawal of growth factors, inappro-

priate growth signals, and viral infection.

The caspase family of aspartate-specific cysteine

proteases has been shown as central executors of ap-

optosis [1,8]. Caspases often function in cascades, in

which an upstream caspase called initiator caspase, suchas caspase-2, -8, -9, and -10, is activated by its interac-

tion with caspase adapter(s). Once activated, the initia-

tor caspase processes and activates one or more

downstream caspases refered to as effector caspases,

such as caspase-3, -6, and -7. The activated effector

caspases then cleave various cellular proteins, leading to

apoptotic cell death. Caspase-3 is the key player and is

activated in a variety of cell types during apoptosis. It is

responsible for the proteolytic cleavage of a set of pro-

teins, such as the nuclear enzyme poly (ADP-ribose)polymerase (PARP) [9].

HDM2 is the human homolog of murine Mdm2 and

is often overexpressed in many human tumors and

cancers including osteogenic sarcomas, soft tissue sar-

comas, gliomas, and breast cancer [10–15]. HDM2 binds

to p53 and inhibits p53-mediated transactivation, G1

arrest, and apoptosis [16,17]. It is known that HDM2

functions as a ubiquitin E3 ligase to target p53 as well asitself for degradation [18–22]. Interestingly, HDM2 has

been shown as a substrate of caspase-3 [23,24]. Caspase-

3 cleaves HDM2 at residue D361 in the DVPD motif,

resulting in the removal of the C-terminal RING finger

domain of HDM2 and stabilization of p53 protein [25].

Biochemical and Biophysical Research Communications 302 (2003) 384–391

www.elsevier.com/locate/ybbrc

BBRC

* Corresponding author. Fax: 1-617-638-5339.

E-mail address: jxiao@bu.edu (Z.-X. Jim Xiao).

0006-291X/03/$ - see front matter � 2003 Elsevier Science (USA). All rights reserved.

doi:10.1016/S0006-291X(03)00189-X

Cleavage of MDM2 has been observed in apoptoticmurine cells [23]. However, HDM2 cleavage can occur

in the absence of apoptosis [25,26].

It is believed that cell cycle and apoptosis are inti-

mately linked and that a coordination and balance be-

tween these two processes are crucial for normal cell

physiology [2,27–29]. The initiation of mitosis during

normal cell cycle in eukaryotic cells requires the acti-

vation of maturation promoting factor (MPF), whichconsists of cdc2 (cdk1) kinase and cyclin B [30]. Inter-

estingly, several studies have shown that cyclin B ex-

pression or cdc2 kinase activity is increased in apoptotic

cells induced by various death signals, such as Fas [31–

34], c-radiation [35], anti-cancer drug 9-nitrocampto-thecin [36], paclitaxel [37], TGF-b [38], or retinoic acid(RA) [39]. However, whether caspase activity is directly

regulated by cyclin-dependent kinase is not entirelyclear.

In this study, we examined HDM2 degradation and

caspase-3 activation in a Xenopus extract system. We

found that HDM2 was specifically cleaved in the

Xenopus mitotic extract due to activated caspase-3 but

not in the interphase extract. In addition, activation of

cdc2 kinase in Xenopus extract system led to caspase-3

activation. Furthermore, ectopic expression of cdc2 ki-nase in HeLa cells also led to activation of caspase-3 and

apoptosis. Our study indicates that deregulation of cdc2

kinase activity can trigger apoptotic machinery that

leads to caspase-3 activation and apoptosis.

Materials and methods

Plasmids, in vitro transcription and translation, and antibodies. The

Bluescript-based plasmids, pHDM2 (1–491), pHDM2 (D220–350),pHDM2 (D220–290), pHDM2 (D220–437) pHDM2 (D361A), andpBluescript/PARP, were provided by Dr. J. Chen (Lee Moffitt Cancer

Center and Research Institute, Tampa, FL). pCDNA3/p53 was

constructed by subcloning the 1.2 kb BamHI–EcoRI fragment of

pGEM4/p53 (provided by Dr. P. Howley, Harvard Medical School,

Boston, MA) into pCDNA3. One lg of plasmid DNA was

transcribed and translated in vitro using TNT Quick Coupled

Transcription/Translation System (Promega) in the presence of 20 lCiLL-[35S]methionine (NEN Life Science Products) at 30 �C for 60min.

Antibodies were purchased that specifically recognize cdc2 (Ab-3,

Oncogene Research Products), cyclin B1 (GNS-1, PharMingen), actin

(C-11 Santa Cruz Biotechnology), and active caspase-3 (D175, Cell

Signaling Technology). An antibody specific for procaspase-3

(29E10B6C10) was a generous gift of Dr. J. Yuan, Harvard Medical

School, Boston, MA.

Mammalian cell culture, transfection, and immunoblotting. Human

cervical carcinoma cell line HeLa (American Type Culture Collection)

was maintained in Dulbecco�s modified Eagle�s medium (DMEM)

supplemented with 10% fetal bovine serum (FBS), 100U penicillin per

ml, 100lg streptomycin per ml, and 2mM LL-glutamine (all materials

purchased from Gibco-BRL) at 37 �C incubator supplemented with 5%CO2. For DNA transfection, cells grown at 70% confluency were

transfected with the indicated plasmid DNA using FuGENE 6

Transfection Reagent (Roche Molecular Biochemicals) and harvested

24 h after transfection. Cells were lysed in lysis buffer (50mM Tris–

HCl, pH 8.0, 125mMNaCl, 0.5% Nonidet P-40, 1mM PMSF, 2lg/ml

leupeptin, and 2lg/ml aprotinin) on ice. Equal amounts of totalprotein (20–50lg) were subjected to electrophoresis on a 10% SDS–

polyacrylamide gel and transferred to PVDF membrane. The mem-

brane was blocked with TBST (25mM Tris–HCl, pH 8.0, 125mM

NaCl, and 0.1% Tween 20) containing 4% non-fat dry milk at room

temperature for 1 h and then incubated with the primary antibody for

1 h at room temperature or overnight at 4 �C. The membrane waswashed three times with TBST and then incubated with a horseradish

peroxidase-conjugated secondary antibody for 1 h at room tempera-

ture. The membrane was then washed four times with TBST. Signals

were detected using Enhanced Chemiluminescense method (ECL,

Amersham Pharmacia Biotech).

Fluorescence activating cell sorting analysis and apoptosis assay.

Cells were trypsinized and washed twice with cold PBS. Cells (1� 106)were subjected to fluorescence-activating cell sorting (FACS) analysis

by FACScan Flow Cytometer (Becton Dickson) [40]. For apoptotic

assay, 1� 105 cells were cytospun onto glass slides (Fisher). Apoptoticcells were detected by In Situ Cell Death Detection Kit (Roche Mo-

lecular Biochemicals). Vectashield mounting medium with propidium

iodide (Vector Laboratories) was used to mount the coverslips onto the

slides. Images were documented under the Olympus IX70 Inverted

Fluorescence Microscope (Spencer Scientific).

Preparation of Xenopus extract and in vitro protein degradation

assay. The Xenopus interphase extracts were prepared according to the

procedure as described [41]. To prepare the Xenopus mitotic extract, a

bacterially expressed non-degradable fragment of sea urchin cyclin B

(D90, 1mg/ml) was incubated with the interphase extract (1:25 ratio) atroom temperature for 1 h [41,42]. One ll of in vitro translated 35S-la-

beled protein was incubated at room temperature with 19 ll of inter-phase or mitotic Xenopus egg extracts, containing 100lg/mlcycloheximide, 1.25mg/ml ubiquitin, and 1� energy mix (7.5mM

creatine phosphate, 1mM ATP, 1mM MgCl2, and 1mM EGTA, pH

7.7). Degradation reactions were terminated at various time points by

mixing with an equal amount of 2� Laemmli�s sample buffer. 35S-la-beled products were separated on 10% SDS–PAGE and detected by

autoradiography. In the degradation assays with inhibitors, MG132

(N-CBZ-Leu-Leu-Leu-al, 1.25mM, Sigma), ALLN (N-Acetyl-Leu-

Leu-Norleu-al, 1.25mM, Sigma), E64 (trans-Epoxysuccinyl-LL-leucy-

lamido (4-guanidino) burane, 1.25mM, Sigma), or caspase-3 inhibitor

Ac-DEVD-CHO (N-Acetyl-Asp-Glu-Val-Asp-AL, 100lM, Sigma)was added to the Xenopus interphase or mitotic extract. To analyze the

role of MPF in the protein degradation assay, 10U of Xenopus re-

combinant p34cdc2/Cyclin B1 (Calbiochem) was pre-incubated with

10 ll of interphase extract for 30min at room temperature prior to theaddition of 35S-labeled proteins.

Histone H1 kinase assay. Xenopus extracts were diluted 50-fold in

the reaction buffer (15mM MgCl2, 20mM EGTA, 10mM dith-

iothreitol, 80mM b-glycerophosphate, pH 7.3, 25lg/ml aprotinin,25 lg/ml leupeptin, 1mM benzamidine, 0.5mM PMSF, and 10 lg/mlpepstatin A). Ten ll of Xenopus extracts was incubated with 6 ll of thereaction buffer in the presence of 1.6mg histone H1 (Calbiochem),

1mM ATP, and 0.25lCi/ll [c-32P]ATP (NEN Life Science Products)

at 30 �C for 15min. Proteins were separated on a 12% SDS–PAGE andthe 32P-labeled histone H1 was detected by autoradiography.

Results

HDM2 is specifically cleaved in Xenopus mitotic extract,

but not in interphase extract

Xenopus egg extract is widely used in the study of

anaphase promoting complex (APC) and APC-depen-

dent protein degradation such as cyclin B [42] and sec-

urin/Pds1 [43]. In Xenopus mitotic extract, cyclin B1 was

L. Gu et al. / Biochemical and Biophysical Research Communications 302 (2003) 384–391 385

specifically degraded (Fig. 1A). We used this system to

study the degradation of HDM2 and p53 at interphase

and mitosis. In vitro translated HDM2 and p53 were

stable in Xenopus interphase extract (Fig. 1B, lanes 1

and 3). However, in mitotic extract, HDM2 protein wascleaved into several small fragments, while p53 protein

remained stable (Fig. 1B, lanes 2 and 4). The major

HDM2 cleavage products were 60, 48, 32 (Fig. 1B, lane

2), and 16 kDa fragments (Fig. 3, lane 2). To investigate

the mechanism of HDM2 cleavage in mitotic extracts,

we used several inhibitors in the reaction mixture, in-

cluding proteosome inhibitors MG132, ALLN, as well

as a cysteine protease inhibitor E-64 [44–46]. As shownin Fig. 1C, none of these inhibitors could effectively

block HDM2 cleavage.

HDM2 is cleaved at D361, a caspase cleavage site

The existence of multiple small fragments generated

in the mitotic extract prompted us to consider the

mechanism of specific protease cleavage. To locate the

cleavage site on HDM2, several deletion mutants ofHDM2 were examined in the in vitro protein degrada-

tion assays. Two internal deletion mutants, HDM2

(D220–290) and HDM2 (D220–350), could be cleaved inmitotic extract (Fig. 2A, lanes 1–4) whereas the deletion

mutant HDM2 (D222–437) was resistant to the cleavage

in mitotic extract (Fig. 2A, lanes 5 and 6). It is noted

that within this region (222–437) there is a caspase-3(CPP32)-like cleavage site at the DVPD sequence after

D361 [23] and that HDM2 protein has been shown to be

cleaved by caspase after D361 during apoptosis, gener-

ating 60 and 30 kDa fragments [23]. To test whether this

D361 residue is involved in the cleavage in mitotic ex-

tract, we examined a point mutant HDM2 (D361A) in

the in vitro protein degradation assay. As shown in Fig.

2B, HDM2 (D361A) could not be cleaved in mitoticextract.

Caspase-3 is activated in Xenopus mitotic extract

Since HDM2 protein is known to be a substrate for

caspase-3 during apoptosis [5,23], we examined whether

caspase-3 is activated in Xenopus mitotic extract. As

shown in Fig. 3, the cleavage of HDM2 in the mitotic

extract in was totally blocked in the presence of thecaspase-3 inhibitor, Ac-DEVD-CHO. In addition, the

Fig. 1. Specific cleavage of HDM2 protein in Xenopus mitotic extract.

(A) 1 ll of in vitro translated 35S-labeled cyclin B1 was mixed with 10llof Xenopus interphase (I) or mitotic (M) extract and incubated at room

temperature for 30min. Five ll of the reaction products was separatedon 10% SDS–PAGE. 35S-labeled products were detected by autoradi-

ography. (B) One ll of in vitro translated 35S-labeled HDM2 (lanes 1

and 2) or p53 (lanes 3 and 4) was mixed with 10 ll of Xenopus inter-phase (I) or mitotic (M) extract and incubated at room temperature for

30min. Five ll of the reaction products was separated on 10% SDS–

PAGE. 35S-labeled HDM2, p53, and the degradation products were

detected by autoradiography. (C) One ll of in vitro translated, 35S-labeled HDM2 was mixed with 10 ll of Xenopus interphase (I) ormitotic (M) extracts in the presence or absence of proteosome inhibi-

tors MG132 (1.25mM), ALLN (1.25mM), or cysteine protease in-

hibitor E64 (1.25mM) at room temperature for 30min. Five ll of thereaction products was separated on 10% SDS–PAGE. 35S-labeled

products were detected by autoradiography.

Fig. 2. HDM2 is cleaved at D361, a caspase cleavage site. One ll of anin vitro translated, 35S-labeled HDM2 deletion mutant (A) or the

caspase cleavage site mutant HDM2 (D361A) (B) was incubated with

10 ll of Xenopus interphase (I) or mitotic (M) extract at room tem-

perature for 30min (A) or for an indicated time (B). Five ll of thereaction products was separated on 10% SDS–PAGE. 35S-labeled

products were detected by autoradiography.

Fig. 3. Activation of caspase-3 in Xenopus mitotic extract. One ll of invitro translated, 35S-labeled HDM2 or N-terminal portion of PARP

was mixed with 10 ll of Xenopus interphase (I) or mitotic (M) extractin the absence or presence of caspase-3 inhibitor Ac-DEVD-CHO

(100 lM) as indicated. The mixture was incubated at room tempera-

ture for 30min. Five ll of the reaction products was separated on 12%SDS–PAGE. 35S-labeled products were detected by autoradiography.

386 L. Gu et al. / Biochemical and Biophysical Research Communications 302 (2003) 384–391

poly (ADP-ribose) polymerase (PARP), a prototypesubstrate of caspase-3 [26], was effectively cleaved in

Xenopus mitotic extract and this cleavage was com-

pletely blocked by the caspase-3 inhibitor Ac-DEVD-

CHO (Fig. 3).

Activation of cdc2 kinase correlates with caspase-3

activity in Xenopus extract

To study the mechanism of caspase-3 activation inXenopus mitotic extract, we first examined the endoge-

nous caspase-3 protein in Xenopus mitotic extract. As

shown in Fig. 4A, as expected, the activated caspase-3 in

the cytochrome c-treated Jurkat cells was detected by an

antibody that can specifically recognize active caspase-3.

The activated caspase-3 was also detected in Xenopus

mitotic extract but not in the interphase extract. Since

the non-degradable form of sea urchin cyclin B

(CYCD90) induces activation of maturation promotingfactor (MPF), we investigated whether there is a corre-

lation between the activation of cdc2 kinase and cas-

pase-3 during interphase to mitosis transition. As shown

in Fig. 4B, HDM2 was cleaved at the time when histone

H1 kinase activity was induced upon addition of

CYCD90 protein, suggesting that CYCD90-inducedcdc2 kinase activation may be important for HDM2

cleavage. We then tested whether a recombinant Xeno-pus p34cdc2/cyclin B (MPF) can result in the HDM2

cleavage. As shown in Fig. 4C, direct addition of MPF

in the interphase extract resulted in HDM2 protein

cleavage, generating the same cleavage products as those

derived from the mitotic extract. These results suggest a

direct link between cdc2 kinase activity and caspase-3

activation.

Ectopic expression of cdc2/cyclin B1 in HeLa cells results

in caspase-3 activation and apoptosis

Given the correlation of activated cdc2 kinase and

caspase-3 activation in Xenopus extract system, we ex-

amined whether ectopic expression of cdc2 kinase can

directly induce caspase-3 activation in mammalian cells.

Activation of caspase-3 in cdc2/cyclin B1 co-transfected

cells was evident by Western blot analysis (Fig. 5A, lane4) whereas single transfection of either cdc2 or cyclin B1

did not lead to a significant increase in activation of

caspase-3 (Fig. 5A, lanes 2 and 3). In addition, FACS

analysis revealed a substantial increase in sub-G1 cell

population in cdc2/cyclin B1 co-transfected cells (Fig.

5B). Furthermore, there were substantially more TU-

NEL positive-stained cells in cdc2/cyclin B1 co-trans-

fected cells (10.8%) than the cells transfected with eithervector (0.7%), cdc2 (1.3%), or cyclin B1 (1.0%) alone

(Fig. 5C). Taken together, these data indicate that

overexpression of cdc2/cyclin B1 can activate caspase-3

and initiate apoptotic pathway in vivo.

Discussion

Xenopus egg extract is widely used in the study of

anaphase promoting complex (APC) and APC-depen-

dent protein degradation [41,42]. Xenopus interphase

extracts supplemented with a non-degradable form of

sea urchin cyclin B (D90) enter and remain in a mitoticstatus, in which maturation promoting factor (MPF) is

activated. The mitotic extract system has been used to

demonstrate the degradation of mitotic APC substrates,such as cyclin B [42] and securin/Pds1 [43].

Cdc2 kinase is required for G2/M transition during

cell cycle. In the interphase Xenopus extract, cdc2 kinase

is low. Cdc2 kinase is activated and maintained at high

level by the addition of a non-degradable cyclin B (D90)into the interphase extract [41,42]. Our collective data

Fig. 4. Correlation of activation of histone H1 kinase activity and

HDM2 cleavage in mitotic extract. (A) Five ll of Xenopus interphase(lane 2) or mitotic (lane 1) extract, 10 ll (�1:5� 106 cells) of Cyto-chrome C-treated (lane 4) or—untreated (lane 3) Jurkat cell extract

(Cell Signaling Technology) was mixed with equal volume of 2�Laemmli�s sample buffer, boiled for 3min, and separated on 10%SDS–PAGE. Protein expression was detected by Western blot anal-

ysis using an antibody specific for active caspase-3 (Cell Signaling

Technology). (B) Non-degradable sea urchin cyclin B D90 protein(1mg/ml) was mixed (at the ratio of 1:25) with Xenopus interphase

extract containing 1ll of in vitro translated, 35S-labeled HDM2. Themixture was incubated for 0, 10, 30, and 60min at room temperature.

Five ll of products was separated on 10% SDS–PAGE. 35S-labeled

products were detected by autoradiography. Kinase assay was per-

formed using histone H1 as the substrate and the 32P-labeled histone

H1 was detected by autoradiography. (C) Ten ll of Xenopus inter-phase extracts containing 1 ll of using in vitro translated, 35S-labeledHDM2 protein was treated with 10U of recombinant Xenopus MPF

at room temperature for 30min. Treated (lane 2) or non-treated (lane

1) extracts were subjected to cdc2 kinase assay using histone H1 as

the substrate and 35S-labeled HDM2 products were detected by au-

toradiography.

L. Gu et al. / Biochemical and Biophysical Research Communications 302 (2003) 384–391 387

suggest that prolonged activation of cdc2 kinase can

trigger the activation of caspase-3. First, activated cas-

pase-3 is readily detected in Xenopus mitotic extract but

not in the interphase abstract. Second, activation of

cdc2 kinase correlates with the activation of caspase-3 as

indicated by the caspase-3 mediated cleavage of HDM2

in Xenopus extract. Third, expression of cdc2/cyclin B1

results in activation of caspase-3 and apoptosis in HeLa

Fig. 5. Overexpression of cdc2/cyclin B1 in HeLa cells results in caspase-3 activation and apoptosis. HeLa cells were either transfected with vector,

cdc2, and cyclin B1 alone, or co-transfected with cdc2 and cyclin B1. Twenty-four hours after transfection, cells were collected. (A) Forty lg of totalprotein extracts was separated on 10% SDS–PAGE and subjected to Western blot analysis using the antibody specific for full-length caspase-3,

activated caspase-3, cdc2, cyclin B1, and actin. (B) Cells (1� 105) were subjected to FACS analysis and percentage of sub-G1 cells was analyzed byCell Quest program. Data shown are representative from two independent experiments. (C) Cells (1� 105) were cytospun onto glass slide. TdT-mediated dUTP nick end labeling (TUNEL) assay was performed to detect apoptotic cells. PI-stained cells (red) and TUNEL-positive cells (green)

were photographed.

388 L. Gu et al. / Biochemical and Biophysical Research Communications 302 (2003) 384–391

cells. Since survivin is a substrate of cdc2 kinase and isimplicated in inhibition of caspase-3 [47,48], it is possi-

ble that abnormal cdc2 kinase may inhibit the survivin-

mediated suppression of caspase-3. However, the exact

mechanism(s) how cdc2 kinase activates caspase-3 must

wait for further investigation.

In an attempt to study the degradation of HDM2 and

p53 at interphase and mitosis, we used the Xenopus egg

extract as a protein degradation assay. It was of interestto note that, unlike p53, which was stable in both in-

terphase and mitotic extract, HDM2 was specifically

cleaved in mitotic extract. The primary cleavage site on

HDM2 is at D361, a caspase-3-dependent cleavage,

since the point mutation of this site and the inhibition of

caspase-3 activity effectively block HDM2 cleavage. The

biological significance of mitotic-specific cleavage of

HDM2 is not clear at this moment. It has been shownthat HDM2 is cleaved at the caspase-3 cleavage site in

both apoptotic [23] and non-apoptotic cells [25,26],

suggesting that cleavage of HDM2 can be dissociated

from apoptosis. Interestingly, p53 has been shown to be

activated at mitosis [49]. Whether HDM2 is cleaved

during normal mitosis and whether this process affects

p53 protein levels or function remain to be determined.

It has been suggested that progression of the cell cycleand control of apoptosis are tightly linked processes. In

fact, cells undergoing mitosis and cells undergoing ap-

optosis share a number of morphological and bio-

chemical features, including rounded cell morphology,

reduction in cell volume, nuclear membrane breakdown,

chromatin condensation, and phosphorylation of lamins

[28]. Although these observations suggest that some of

the biochemical changes during these two processesmight be similar, whether activation of caspases is in-

volved in the biochemical and morphological changes

during normal mitosis is not clear. It is conceivable that,

under normal physiological condition, cdc2 kinase reg-

ulates caspases in such a way that the temporal and

spatial activation on the caspase activity restricts casp-

ases to only mitotic processes but does not trigger ap-

optosis.It has been shown that increased cdc2 kinase activity

is linked to multiple forms of apoptosis induced by

several death signals. For instance, Fas-induced apop-

tosis requires cdc2 kinase [32–34]. Expression of cyclin

B1 and/or cdc2 has been found upregulated in apoptotic

cells treated by c-irradiation [35], camptothecin [36],paclitaxel [37], or TGFb-1 [38]. Interestingly, it has beendemonstrated that Fragmentin induces premature acti-vation of cdc2 kinases, resulting in apoptosis [50]. On

the other hand, TNF-a-induced apoptosis is inhibited bythe expression of a dominant-negative cdc2 mutant [51].

These data support the notion that cdc2 kinase activity

plays an important role in apoptotic program and that

improper activation of cdc2 kinase can trigger apopto-

sis. Consistent with this notion, our data indicate that

forced expression of cyclin B1 and cdc2 results in ap-optosis, likely due to the activation of caspase-3.

Abnormal expression of various cyclins and cyclin-

dependent kinases has been shown to be associated with

many types of human cancers [52,53]. Cyclin D1 and

cyclin E are frequently overexpressed and dysregulated

in a variety of human cancers. For instance, overex-

pression of cyclin D1 occurs in 60% of breast cancers,

40% of colorectal cancers, 40% of squamous carcino-mas, and 20% of prostate cancers [52–55]. However,

overexpression of cyclin B or cdc2 kinase has rarely been

observed in human tumors and cancers. It is conceivable

that there is a selective pressure against cells with ab-

normal cdc2 kinase as it induces apoptosis.

Acknowledgments

We thank Dr. Y. Wan (Harvard Medical School) for Xenopus

extract and valuable advice for in vitro protein degradation assays, Dr.

J. Chen (Lee Moffitt Cancer Center and Research Institute, Tampa,

FL) for plasmids, and Dr. J. Yuan (Harvard Medical School) for the

antibody. We also thank Dr. Y. Zhang to assist in FACS analysis. This

work was supported by NIH/National Cancer Institute Grant

R01CA79804 and the Department of Defense Breast Cancer Research

Grant DAMA17-97-1-7311 (both to Z.X.X.)

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