Antitumor activity of fludarabine against human multiple myeloma in vitro and in vivo

Antitumor activity of fludarabine against human multiplemyeloma in vitro and in vivoHaitao Meng1, Chunmei Yang1, Wanmao Ni1, Wei Ding1, Xiudi Yang1, Wenbin Qian1,2

1Institute of Hematology, the First Affiliated Hospital, College of Medicine, Zhejiang University; Key lab of Combined Multi-Organ Transplantation,

Ministry of Public Health, Hangzhou; 2Xinyuan Institute of Medicine and Biotechnology, School of Life Sciences, Zhejiang University of Technology

and Sciences, Hangzhou, China

Multiple myeloma (MM) is an incurable B-cell malig-

nancy resulting in significant morbidity and mortality

(1). MM accounts for 1% of all cancers and slightly

more than 10% of hematological malignancies. Although

many therapeutic advances such as combined chemo-

therapy and hematopoietic stem cell transplantation have

been made to improve the survival rate of patients with

MM, a higher proportion of the patients cannot expect

the long-term remission due to drug-resistant disease,

and minimal residual disease, leaving limited therapeutic

option (2). Therefore, new approaches to therapy are

required.

Fludarabine, an arabino-adenosine analogue, inhibits

DNA synthesis in proliferating cells; however, in indolent

cells fludarabine has an inhibitory effect on RNA tran-

scription (3), thus playing a major role in the treatment

of B-cell lymphocytic leukemia, hairy cell leukemia, and

indolent lymphomas. Because MM cells proliferate very

slowly, it is plausible that inhibition of RNA synthesis

may be an important growth inhibitory effect in MM

cells. In fact, the in vitro anticancer activity of fludara-

bine against MM cell lines is demonstrated (4). Although

previous results of phase II clinical trials in MM with

fludarabine were completely negative (5, 6), recent stud-

ies show that the treatment of fludarabine combined with

other chemotheraputic agents is clinically effective in the

patients with newly diagnosed and relapse or refractory

MM (7, 8). Moreover, 8-amino-adenosine (a novel pur-

ine analogue) and gemcitabine have been demonstrated

to be effective in the induction of apoptosis in myeloma

Abstract

Fludarabine, a nucleoside analogue, plays a major role in the treatment of B-cell lymphocytic leukemia,

hairy cell leukemia, and indolent lymphomas. There is a controversy about antitumor activity of fludarabine

in multiple myeloma (MM). The aim of this study was to evaluate the activity of fludarabine against human

myeloma cells both in vivo and in vitro. We demonstrated that myeloma cell line RPMI8226 was efficiently

inhibited by fludarabine, concomitantly with decreased phosphorylation of Akt, down-regulation of the

inhibitor of apoptosis proteins (IAP) family, including XIAP and survivin, and induction of apoptosis related

to activation of caspase cascade. Contrary to dexamethasone, the effect of fludarabine on RPMI8226 cells

was independent of interleukin-6. Fludarabine also induced cytotoxicity in dexamethasone-sensitive

(MM.1S) and -resistant (MM.1R) cells at 48 h with IC50 of 13.48 lg ⁄ mL and 33.79 lg ⁄ mL, respectively. In

contrast, U266 cells were resistant to fludarabine. Moreover, RPMI8226 myeloma xenograft model was

established using severe combined immunodeficient mice. The tumors treated with fludarabine at

40 mg ⁄ kg increased less than 5-fold in 25 d comparing with approximately 10-fold in the control tumors,

demonstrating the antitumor activity of fludarabine in vivo. These results suggest that fludarabine may be

an important therapeutic option for MM patients who are resistant to dexamethasone.

Key words multiple myeloma; fludarabine; apoptosis

Correspondence W. Qian, Institute of Hematology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Qingchun

Road 79, Hangzhou, 310003, China. Tel: 86-571-56723008; Fax: 86-571-87236702; e-mail: [email protected]

Accepted for publication 15 August 2007 doi:10.1111/j.1600-0609.2007.00968.x

ORIGINAL ARTICLE

European Journal of Haematology ISSN 0902-4441

486ª 2007 The Authors

Journal compilation 79 (486–493) ª 2007 Blackwell Munksgaard

(9–11). These data provide an impetus to evaluate the

role of fludarabine in MM.

In this study, we examined the effects of fludarabine

on MM cell lines, and studied the mechanisms of cyto-

toxicity of fludarabine in myeloma cells with attention to

the alteration of survival pathways, including Akt and

the inhibitor of apoptosis proteins (IAP) family. Further-

more, we also evaluated the potent antitumor activity of

fludarabine in human myeloma RPMI8226 xenograft

mice model.

Materials and methods

Cell lines, cultures, and reagents

Dexamethasone-sensitive (MM.1S) and -resistant

(MM.1R) human MM cell lines were kindly provided by

Steven Rosen (Northwestern University, Chicago, IL,

USA). RPMI8226 and U266 cell lines were purchased

from American Type Culture Collection (Rockville, MD,

USA). Cells were cultured with RPMI 1640 (Hyclone

Laboratories, Logan, UT, USA) supplemented with 5%

fetal bovine serum (Hyclone Laboratories), 1% l-gluta-

mine, and 0.1% gentamycin, and maintained at 37 �C in

an incubator with 5%CO2. Reagents were purchased

from the following vendors: fludarabine phosphate

(Schering, Germany); interleukin-6 (IL-6; Upstate, New

York, NY, USA); pan-caspase inhibitor zVAD-FMK

(BioVision, Mountain View, CA, USA); dexamethasone

(Sigma, St Louis, MO, USA).

Growth inhibition assay

The inhibitory effect of fludarabine on proliferation of

MM cell lines was assessed by the 3-(4,5-dimethylthiazol-

2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma)

assay as described previously (12).

Cell-cycle analysis and apoptosis determination

Cells (5 · 105 cells) were washed twice in phosphate-

buffered saline (PBS) and fixed with 70% ice-cold

ethanol, then centrifuged and suspended in PBS

containing 100 lg ⁄mL RNase A. After incubated for

30 min at 37�C, samples were resuspended in

25 lg ⁄mL propidium iodide (Sigma). Flow cytometry

was performed on a FACSCalibur automated system

(Becton Dickinson, Franklin Lakes, NJ, USA). Apop-

tosis was determined by Annexin V-FITC apoptosis

detection kit (BD Pharmingen, SanDiego, CA, USA),

according to the manufacturer’s instructions. For

TUNEL (terminal deoxynucleotidyl transferase-

mediated deoxyuridine triphosphate nick end labeling)

assay, cells were analyzed by flow cytometry using the

in situ cell death detection kit (Roche, Philadelphia,

PA, USA), according to the manufacturer’s

instructions.

Mitochondrial membrane potential measurement

PRMI8226 cells treated with fludarabine were washed

and stained with 5 lg ⁄mL of Rhodamine 123 (Molecu-

lar Probes, Eugene, OR, USA) at 37�C for 30 min.

Rhodamine 123 was excited with a 488 nm argon ion

laser; fluorescence emission was measured at 530 nm

using flow cytometry.

Western bolts

Western blots were done as previously described (12).

The primary antibodies used here were as follows: poly

(adenosine diphosphate-ribose) polymerase (PARP), cas-

pase-8, -9, -7, -3, Bax, Bid, Bak, Survivin, XIAP, and

cIAP were purchased from Cell Signaling Technology

(Beverly, MA, USA); Rabbit monoclonal antibodies to

total Akt, Ser 473 phosphorylated Akt, and GAPDH

were provided by Kangchen (Shanghai, China); Mono-

clonal second mitochondrial-derived activator of caspase

(Smac) antibody and b-actin were purchased from Santa

Cruz Biotechnology (Santa Cruz, CA, USA).

Establishment of subcutaneous and disseminated MMxenografts and therapy

Severe combined immunodeficient (SCID) mice (Shang-

hai Experimental Animal Center of the Chinese Acad-

emy of Sciences, China) were housed and maintained

in facilities under an institute-approved animal proto-

col. For the s.c. xenograft MM RPMI 8226 mouse

model, 3- to 4-wk-old female mice were inoculated

subcutaneously with 10 · 106 RPMI 8226 cells. When

tumor volumes approached 100 mm3, the mice were

divided into experimental cohorts of six mice each.

Injections (i.p.) of fludarabine or PBS (control) were

administered each day for 3 d. Tumor volume was cal-

culated by using the formula: 4p ⁄3 · (tumor

width ⁄ 2)2 · (tumor length ⁄ 2) described as previously

(13).

Immunohistochemistry

Seven days after treatment of fludarabine, some addi-

tional mice were humanely killed with CO2, and the

tumor mass was bisected using a razor blade. The

tumors were immediately placed in 10% buffered formal-

dehyde overnight. Formaldehyde-fixed tumors were

embedded in paraffin and cut into 4 lm-thick serial

sections using standard histologic procedures.

Meng et al. Antitumor activity of fludarabine against multiple myeloma

ª 2007 The Authors

Journal compilation 79 (486–493) ª 2007 Blackwell Munksgaard 487

Statistical analysis

Tumor volumes were calculated as mean ± SEM. The

statistical of experimental results was calculated by

one-way analysis of variance (anova) and Student’s

t-test. P < 0.05 considered to be significant.

Results

Treatment with fludarabine reduced myeloma cellproliferation independent of interleukin-6

The effect of fludarabine on growth of MM cell lines

was determined using MTT assays. Fludarabine inhibited

efficiently the proliferation of RPMI 8226 cells in a

dose–time-dependent fashion, with 50% inhibition (IC50)

at 24 h of 1.54 lg ⁄mL (Fig. 1A). The IC50 of MM.1S

and MM.1R cells at 48 h was 13.48 lg ⁄mL and

33.79 lg ⁄mL, respectively (Fig. 1B). In contrast, U266

cells were resistant to fludarabine with IC50 of

222.2 lg ⁄mL at 48 h (data not shown). Because IL-6 acts

as a growth factor for MM cells (14), we evaluated the

effect of fludarabine on RPMI 8226 in the presence of

exogenous IL-6. IL-6 (50 ng ⁄mL) did not provide protec-

tion against fludarabine-induced growth inhibition and

apoptosis (data not shown). We next examined whether

fludarabine could enhance the effect of dexamethasone

on MM1.S cell line. The results showed that fludarabine

enhanced significantly the effect of dexamethasone

(P < 0.01; Fig. 1C).

Inhibition of myeloma cell proliferation was due to G1arrest and apoptosis induction

We next did cell cycle analysis using propidium iodide-

staining and subsequent flow cytometry sorting. As

depicted in Fig. 2A, fludarabine treatment resulted in

increased number of cells in the G1 phase of cell cycle,

accompanied with a concomitant reduction of cells at the

S phase of cell cycle in a time-dependent manner. The

apoptotic nature of cell death induced by fludarabine

was further confirmed by Annexin-V binding (Fig. 2B),

and TUNEL assay (Fig. 2C). These results indicate that

fludarabine induces a cell cycle block and triggers apop-

tosis in MM cells.

Apoptosis triggered by fludarabine is mediated viacaspase-3, caspase-7, and PARP cleavage

We further used immunoblotting to assess activation of

caspases in RPMI 8226 cells. As shown in Fig. 3A,

fludarabine triggered time-dependent cleavage of caspase-

8, -9, and -3, -7, followed by PARP cleavage. The pan-

caspase inhibitor zVAD-FMK (30 lm) resulted in an

almost 50% reduction of apoptosis (data not shown),

suggesting that caspase activation is involved in the

mechanism of fludarabine-induced apoptosis.

Previous studies suggest the involvement of bcl-2

family protein in myeloma cell death (15, 16). We found

that fludarabine induced increased expression of Bax in a

0 8 16 64 µg/mL32

A B

00

20

40

60

% V

iabi

lity

Via

bilit

y (%

)

Via

bilit

y (%

)

80

0

20

10

40

30

60

50

80

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12024 h48 h

MM.1S; 24 hMM.1S; 48 hMM.1R; 24 hMM.1R; 48 h

0

20

40

60

80

100

120

0.5 2 4 µg/mL1

C

Dex 1 µM Flud 8 µg/mL Dex +Flud 4 µg/mL

Dex +Flud 8 µg/mL

Flud 4 µg/mL

P < 0.01P < 0.01

***

P < 0.01P < 0.01

***

Figure 1 Fludarabine reduces the viability of

multiple myeloma cell lines. The cells were cul-

tured with indicated concentration of fludarabine

for 24 and 48 h. Cell viability was measured by

MTT assay. (A) RPMI8226. (B) MM.1S and

MM.1R. (C) Synergistic effects of fludarabine

with dexamethasone. MM.1S cells were treated

with dexamethasone (1 lM) or fludarabine at

4 lg ⁄ mL and 8 lg ⁄ mL alone, and combination

of two agents for 24 h. Bars represent mean ±

SEM, n = 3. *P < 0.01 vs. fludarabine;

**P < 0.01 vs. dexamethasone.

Antitumor activity of fludarabine against multiple myeloma Meng et al.



time-dependent fashion, while the expression of Bak

didn’t change (Fig. 3B). Importantly, decreased expres-

sion of Bid, a bcl-2 family member which cooperates

with Bax to cause mitochondrial dysfunction (17), was

triggered by fludarabine (Fig. 3B), suggesting that cross-

talk of apoptotic signaling from caspase-8 to caspase-9

occurs. After exposure to fludarabine for 12 h, RPMI

8226 cells showed a loss of membrane potential with

61.05% of the cells expressing low fluorescence of rhoda-

mine 123 compared with 8.62% of cells in untreated con-

trol (data not shown). Finally, the release of Smac was

also observed in response to this agent (Fig. 3B).

Alterations of survival pathways are associated withfludaranine-mediated apoptosis

Because Smac reportedly promotes apoptosis by binding

to and antagonizing the IAP family multiple proteins

including XIAP, cIAPs, and survivin (18), the expression

of these genes was examined. Downregulation expression

of XIAP and survivin was observed in fludarabine-

treated RPMI8226 cells, whereas expression of cIAP did

not change (Fig. 4A). Furthermore, decreased expression

of survivin was also observed in MM.1S, and MM.1R,

but not in U266 cells that is resistant to fludarabine

(Fig. 4B).

S 65%

6 h

G0/G1 68%Sub-G1 27%S 32%

PI

Control

Control 6 h 2 h 24 h

dUTP-Fluos

Counts

Annexin-FITC

G0/G1 27%Sub-G1 1%R

PM

I 8226

ControlA

B

C

24 h12 h

G0/G1 42%Sub-G1 12%S 53%

3.65% 6.44% 58.15% 67.76%

G0/G1 53%Sub-G1 25%S 46%

1 µg 2 µg 4 µg

Figure 2 Treatment of fludarabine results in

G1-arrest and apoptosis of myeloma cells. (A)

RPMI8226 cells were treated with fludarabine

(2 lg ⁄ mL) for indicated times. Cell cycle analy-

sis was done after propidium iodide staining.

(B) RPMI 8226 cells incubated with indicated

concentration of fludarabine for 24 h, stained

with FITC-conjugated Annexin V and propidium

iodide, and then subjected to flow cytometric.

(C) RPMI 8226 cells were treated with 2 lg ⁄ mL

fludarabine for the indicated times. Apoptotic

cells were evaluated by TUNEL assay. A repre-

sentative of three separate experiments is

shown.

A

B

Figure 3 Apoptosis triggered by fludarabine is mediated via caspase

cascade. (A) Activation of caspases. (B) Expression of bcl-2 family pro-

tein. Cells were treated with fludarabine (2 lg ⁄ mL) for the indicated

times. Whole cell extracts were prepared and immunoblotted. The

data are representative of two determinations with identical results.


ª 2007 The Authors


We next examined expression of Akt. The phosphory-

lation of Akt at position Ser 473 was highly expressed in

MM.1R, MM.1S, and RPMI8226, but lower in U266

cells that are resistant to fludarabine (Fig. 4C). Fludara-

bine inhibited phosphorylation of Akt in some, but not

all, myeloma cell lines (Fig. 4D), reflecting a high degree

of heterogeneity. We therefore evaluated the contribution

of this pathway in fludarabine-mediated apoptosis.

Apoptosis assays were done on myeloma cell lines trea-

ted with the Akt inhibitor (30 lm LY294002) in the pres-

ence or absence of fludarabine (2 lg ⁄mL). LY294002

slightly induced the apoptosis of RPMI8226 and not of

U266 cells. Interestingly, pronounced apoptosis of

MM.1S and MM.1R cells were observed (Fig. 4E). Slight

potentiation was observed in all of tested cell lines except

U266 when LY294002 (30 lm) and fludarabine

(2 lg ⁄mL) were added together (Fig. 4E).

Fludarabine induces apoptosis and significantlyinhibits myeloma cell growth in vivo

We also addressed the issue of whether fludarabine could

inhibit tumor growth in vivo. The results showed that

control tumors treated with PBS grew rapidly to approx-

imately 10-fold their initial volume in 25 d, whereas, the

tumors in the fludarabine at 40 mg ⁄kg increased less

than 5-fold. A significant (P < 0.05) antitumor effect of

40 mg ⁄kg fludarabine on RPMI8226 tumor growth was

demonstrated (Fig. 5A). In contrast, fludarabine at

8 mg ⁄kg had no effect (P > 0.05). TUNEL assay

showed an evident increase in apoptotic nuclei in

RPMI8226 tumors treated with 40 mg ⁄kg fludarabine at

day 10 (Fig. 5B). Together, these data suggest that flu-

darabine is effective in suppressing RPMI8226 myeloma

xenografts in SCID mice.

Discussion

Fludarabine, combined with VAD regimen (19), has been

used for treating newly diagnosed MM patients (7). Of

the ten patients, nine responses at 1 month post-therapy

follow-up with two in complete remission (CR) and

seven patients in partial remission (PR). In contrast, of

the nine patients treated with VAD alone, five responses

in PR. In a clinical investigation reported by Luo et al.

(8), 11 heavily pretreated MM patients receive two cycles

of fludarabine combined with mitoxantrone and

dexamethasone. The response rate was 45.5% (five PR),

compared with 22.7% in the VAD regimen. Here, we

show that RPMI8226, MM.1S, and MM.1R myeloma

cell lines are blocked in G1 phase of the cell cycle and

undergo apoptosis when treated with pharmacological

concentrations of fludarabine (20). The activation of

both caspase-8 and -9 followed by downstream activa-

tion of caspase-3, -7, and PARP is observed in fludara-

bine-treated MM cells. Those results are supported with

previous reports showing that most conventional

Control 6 12 24 h

MM.1R

MM.1R

MM.1R

MM.1R

Control 24 h Control 24 h Control 24 h Control 24 h

survivin

0 6 0 6 0 6 0 6 h

16Kd

36Kd

36KD

60kD

60kD

36KDa

60kDa

60kDa

GAPDH

GAPDH

GAPDH

80

70

60

50

40

Apo

ptot

ic c

ells

(%

)

30

20

10

0

pAKT

pAKT

tAKT

tAKT

MM.1S

MM.1S

MM.1S

MM.1S

RPMI8226

RPMI8226

RPMI8226

RPMI8226

Con Ly Flud L+F Con Ly Flud L+FCon Ly Flud L+FCon Ly Flud L+F

U266

U266

U266

U266

A

B

C

D

E

Figure 4 Effects of fludarabine on survival pathways. (A) RPMI8226

cells were treated with fludarabine (2 lg ⁄ mL) for the indicated times

and cell lysates were analyzed by immunoblotting for the expression

of IAPs proteins. (B) MM cell lines were incubated with fludarabine at

2 lg ⁄ mL for 6 h. Immunoblotting was performed. (C) The whole cell

lysates from RPMI8226, U266, MM.1S, and MM.1R myeloma cell line

were examined using immunoblotting with specific antibodies against

Akt and phospho-Akt (at Ser473). (D) Myeloma cells were cultured

with 2 lg ⁄ mL fludarabine for 24 h and cell lysates were subjected to

western blot analysis. (E) The combined effects of fludarabine and Akt

inhibitor LY294002. Myeloma cell lines were cultured with fludarabine

(2 lg ⁄ mL) or LY294002 (30 lM), and combination of two agents for

36 h, and then analyzed by flow cytometry for apoptosis. The results

are representative of three independent experiments. Bars represent

mean ± SEM, n = 3.




including dexamethasone, and novel chemotherapeutic

agents trigger the apoptosis of MM cell via activation of

caspase-8 (15, 21, 22) or caspase-9 (10, 23). In our mye-

loma model, increased expression of Bax, decreased

mitochondrial membrane potential, and release of Smac

occur. These data suggest that two main pathways of

procaspase activation (intrinsic mitochondrial pathway

and extrinsic death receptor pathway) are involved in flu-

darabine-induced apoptosis of myeloma cell.

IL-6 triggers proliferation and survival of MM cells

via activation of multiple signal pathways (24, 25). Spe-

cifically, dexamethasone resistance conferred by IL-6 is

mediated via the phosphatidylinositol-3 kinase (PI3-

K) ⁄Akt signaling cascade (14, 26). Our data indicate that

IL-6 does not abrogate fludarabine-induced apoptosis of

RPMI8226, consistent with a previous report showing

that the cytotoxicity of gemcitabine against MM cells is

independently of IL-6 (11). In addition, we show that flu-

darabine enhances significantly the effect of dexametha-

sone on MM.1S cells. Collectively, fludarabine, contrary

to dexamethasone, have the pathways bypassing IL-6,

and therefore might become an important therapeutic

option for MM patients who are resistant to dexametha-

sone.

The Akt kinase is a well-studied viability-promoting

effector molecule that is activated downstream of PI3-K.

Recently, activated Akt kinase has been demonstrated in

MM cell lines (26) and patient’s tumors (27). Our preli-

minary data show a different degree of activation level

of Akt in all tested MM cell lines. Interestingly, Akt

inhibitor LY294002 produces a much stronger cytotoxic-

ity in MM.1S and MM.1R that have highly phosphory-

lated Akt at Ser473, compared with RPMI8226 cells that

contained weak activation of Akt. In contrast, U266 cells

are resistant to LY294002 or fludarabine due to its low-

est phosphorylation of Akt. These data suggest that the

activation of Akt correlates with the sensitivity of MM

cells to AKT inhibitor, and is involved in fludarabine-

induced myeloma cell death.

XIAP inhibits apoptosis targeting the effector caspase-

3 and -7, as well as the initiator caspase-9 (28–30). Fur-

thermore, XIAP may play a role in worsening the prog-

nosis of MM patients in association with chemotherapy-

induced overexpression of multidrug-resistant protein or

lung resistance protein (31). Our data show that the

expression of XIAP was decreased during fludarabine-

induced apoptosis. Survivin is also a fascinating member

of IAP family with its dual roles in mitosis and apoptosis,

Control

00

200

400

600

800

Tum

or V

ol (

mm

3 )

1000

1200

1400ControlFludarabine (8mg/kg)Fludarabine (40mg/kg)

7 10 13 16 19 22 25 Days

Fludarabine

*

A

BFigure 5 Antitumoral efficacy of fludarabine in

established RPMI8226 tumors in vivo. (A)

RPMI8226 human myeloma xenografts implanted

in severe combined immunodeficient mice. PBS

or fludarabine were injected intraperitoneally on

three consecutive days. Tumor growth of the

mice was measured at the indicated times. Data

are presented as the tumor mean volume ± SE.

*P < 0.05 vs. control without fludarabine

(Student’s t-test). (B) Tumor sections were

excised and analyzed for induction of apoptosis

by TUNEL staining. Arrows denote cells under-

going apoptosis. Original magnification, ·40.


ª 2007 The Authors


and emerges as an attractive target for cancer therapy

(32). A significant correlation between survivin expression

at protein level and clinical course of MM is demon-

strated (33). Moreover, survivin knockdown by RNA

interference leads to growth inhibition, apoptosis, and

enhanced sensitivity of myeloma cell to conventional

anti-myeloma agents (33). We demonstrate that the

action of fludarabine correlates to the downregulation of

survivin. Altogether, cellular cytotoxicity of fludarabine

in myeloma cells appears to be mediated through multiple

pathways, although the precise pathways and exact mech-

anisms involved in apoptotic cell death are still unknown.

In summary, this study provides evidence that fludara-

bine induces significant cytotoxicity in dexamethasone-

sensitive and -resistant myeloma cell lines in vitro. The

mechanisms of fludarabine cytotoxicity are related to cell

cycle arrest and induction of apoptosis, which appears to

be regulated by multiple pathways including Akt and

IAP family, and activation of caspases. Moreover, in vivo

antitumor activity of fludarabine is demonstrated in mye-

loma xenograft model. We conclude that fludarabine

may be clinically effective in a subset of MM patients,

but responders cannot be easily pre-selected on the basis

of either the conventional clinical and pathologic

characteristics.

Acknowledgements

This work was supported by a grant from Science and

Technology of Zhejiang Province, China (2005c23017).

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ª 2007 The Authors


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Antitumor activity of fludarabine against human multiple myeloma in vitro and in vivo