Phosphoinositide 3-kinase/Akt inhibition increases arsenic trioxide-induced apoptosis of acute...

Phosphoinositide 3-kinase/Akt inhibition increases arsenictrioxide-induced apoptosis of acute promyelocytic and T-cellleukaemias

Acute promyelocytic leukaemia (APL) is a rare disease

representing 10–15% of all adult acute myeloid leukaemia

(AML) patients. APL is characterised by developmental arrest

of granulopoiesis at the promyelocytic stage and is generally

associated with a specific t(15;17) chromosomal translocation,

which fuses the PML protein and retinoic acid receptor (RAR)

genes to yield a PML–RAR fusion protein (Puccetti &

Ruthardt, 2004). Arsenic trioxide (As2O3) is a clinically

effective agent in the treatment of APL. At clinically achievable

concentrations (1–5 lmol/l in the plasma, see Evens et al,

2004; Douer & Tallman, 2005), As2O3 provokes the degrada-

tion of the PML-RAR oncogenetic fusion protein, expressed in

the vast majority of APLs, thus forcing the malignant

promyelocytes to terminal differentiation and/or apoptosis

(Zhu et al, 2002). Currently, As2O3 is considered the treatment

of choice for patients with relapsed disease, particularly in

patients exposed to retinoids within the previous 12 months

(Soignet et al, 2001). Furthermore, albeit with lower efficacy,

As2O3 also induces apoptosis in tumour cells lacking the

PML-RAR, including other types of leukaemia and multiple

Giovanna Tabellini,1 Pier Luigi Tazzari,2

Roberta Bortul,3 Camilla Evangelisti,4

Anna Maria Billi,4 Tiziana Grafone,5

Giovanni Martinelli,5 Michele Baccarani5

and Alberto M. Martelli4,6

1Dipartimento di Scienze Biomediche e

Biotecnologie, Sezione di Citologia e Istologia,

Universita di Brescia, Brescia, 2Servizio di

Immunoematologia e Trasfusionale, Policlinico

S.Orsola-Malpighi, Bologna, 3Dipartimento di

Morfologia Umana Normale, Universita di

Trieste, Trieste, 4Dipartimento di Scienze

Anatomiche Umane e Fisiopatologia

dell‘Apparato Locomotore, Sezione di Anatomia,

Cell Signalling Laboratory, Universita di Bologna,

Bologna, 5Istituto di Ematologia ed Oncologia

Medica Seragnoli, Universita di Bologna, Bologna,

and 6Istituto per i Trapianti d’Organo e

l’Immunocitologia del C.N.R., Sezione di Bologna

c/o I.O.R., Bologna, Italy

Received 01 May 2005; accepted for publication

21 June 2005

Correspondence: Alberto M. Martelli, MD,

Dipartimento di Scienze Anatomiche Umane e

Fisiopatologia dell’Apparato Locomotore,

Universita di Bologna, via Irnerio 48, 40126

Bologna, Italy.

E-mail: [email protected]

Summary

Recent studies suggest that the prosurvival signal transduction pathway

involving phosphoinositide 3-kinase (PI3K)/Akt can confer an aggressive,

apoptosis-resistant phenotype to acute leukaemia cells. We have investigated

the effect of modulating this signalling pathway on the sensitivity of

leukaemic cell lines (NB-4, CEM, Jurkat, MOLT-4) and acute promyelocytic

primary blasts to apoptosis induced by 1 lmol/l As2O3. Whereas parental

NB-4 cells did not display any phosphorylated (active) Akt, CEM, Jurkat and

MOLT-4 cells exhibited high levels of Akt activation. Consistently, treatment

of NB-4 cells with pharmacological inhibitors of the PI3K/Akt pathway

(LY294002, wortmannin) did not increase sensitivity of these cells to arsenic

trioxide (As2O3), whereas siRNA knock-down of Akt enhanced As2O3-

induced apoptosis of CEM, Jurkat and MOLT-4 cells. Overexpression of a

constitutively active Akt cDNA rendered NB-4 cells less susceptible to As2O3.

Upon prolonged exposure to As2O3, we isolated a NB-4 cell clone that was

resistant to As2O3 and displayed high levels of active Akt. LY294002

treatment of acute promyelocytic primary blasts with elevated Akt

phosphorylation levels resulted in an increased sensitivity to As2O3. These

results may provide a rationale for the development of combined or

sequential treatment with PI3K/Akt inhibitors to improve the efficacy of

As2O3 on acute leukaemias and also to overcome As2O3 resistance.

Keywords: apoptosis, leukaemia, protein phoshorylation, signal transduc-

tion.

research paper

doi:10.1111/j.1365-2141.2005.05679.x ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 130, 716–725

myeloma, which opens the possibility for broader clinical

application of this compound (Miller et al, 2002). For

example, As2O3 has been recently tested for the treatment of

acute T-cell leukaemia in combination with interferon

(Bazarbachi et al, 2004). Moreover, murine T-cell prolympho-

cytic leukaemia responds to As2O3 treatment in vitro and in

vivo (Recher et al, 2002). Nevertheless, it would be advisable to

elucidate strategies to increase the apoptotic action of As2O3

and reduce drug dosage. An important aspect of apoptosis

regulation is the signalling by serine/threonine kinases, a broad

category of kinases that includes, among others, the extracel-

lular-regulated protein kinases 1/2 (Erk 1/2) and Akt (Cross

et al, 2000). To be activated, Akt requires the 3¢ phosphory-

lated lipid products of phosphoinositide 3-kinase (PI3K)

(Hanada et al, 2004). Indeed, 3¢-phosphoinositides attract to

the plasma membrane phosphoinositide-dependent kinase 1

(PDK1) and Akt. PDK1 activates Akt by phosphorylation of

Thr 308 residue in the activation loop, although full activation

requires phosphorylation of Ser 473 in the C-terminal

hydrophobic motif of Akt. The latter phosphorylation step is

effected by a kinase that has not been conclusively identified so

far (Brazil et al, 2004; Hanada et al, 2004). The signalling

pathways that emanate from Erk 1/2 and PI3K/Akt are

considered very critical for cell survival to apoptotic stimuli

(Hanada et al, 2004; Wada & Penninger, 2004). For this

reason, these two signalling pathways represent promissory

targets of therapeutic intervention in haematological malig-

nancies (Platanias, 2003; Martelli et al, 2005). Indeed, recent

studies have highlighted that both Erk 1/2 and PI3K/Akt

pathways can be constitutively activated in AML blasts, and

their down-modulation by means of selective pharmacological

inhibitors induces apoptosis and/or increases blast sensitivity

to chemotherapeutic agents (Milella et al, 2001; Lunghi et al,

2003; Xu et al, 2003; Bortul et al, 2005). It has been shown that

inhibition of the Erk 1/2 pathway synergises with As2O3 to

induce apoptosis in APL cells (Lunghi et al, 2005). Moreover,

we have recently demonstrated that down-modulation of the

PI3K/Akt pathway increased sensitivity to As2O3 of a HL60 cell

clone with a constitutively activated PI3K/Akt axis (Tabellini

et al, 2005). Although HL60 cells are sensitive to retinoids,

which promote their granulocytic differentiation, they are not

representative of a typical APL, as they lack the t(15;17)

chromosomal translocation that is a distinctive feature of APL.

In this study, we investigated whether or not down-modula-

tion of PI3K/Akt pathway could potentiate the antileukaemic

action of As2O3 in cells from APL and acute T-leukaemia. We

found that pharmacological inhibitors of the PI3K/Ak signal-

ling network restored As2O3 sensitivity of a NB-4 cell clone

with an upregulated PI3K/Akt axis that was resistant to As2O3.

Down-modulation of Akt phoshorylation also significantly

enhanced As2O3-induced apoptosis of primary APL blasts.

Finally, blocking PI3K/Akt function with specific double-

stranded RNA oligonucleotides (siRNA) increased As2O3

sensitivity of T-leukaemia cell lines, which have constitutively

active Akt signalling.

Materials and methods

Chemicals and antibodies

As2O3, normal goat serum (NGS), bovine serum albumin (BSA,

Fraction V), monoclonal antibody to tubulin and peroxidase-

conjugated secondary antibodies were from Sigma (St Louis,

MO, USA). The COMPLETE Protease Inhibitor Cocktail,

Annexin V-fluorescein isothiocyanate (FITC) staining kit, and

the Lumi-LightPlus enhanced chemiluminescence (ECL) detec-

tion kit were from Roche Applied Science (Milan, Italy).

Wortmannin and LY294002 were from Calbiochem (La Jolla,

CA, USA). The In situ Cell Death Detection kit for TUNEL (TdT-

mediated dUTP Nick End Labelling) was from Roche Applied

Science. The Protein Assay kit (detergent compatible) was

from Bio-Rad (Hercules, CA, USA). Constitutively active and

dominant negative Akt cDNAs (both cloned in pUSEamp and

c-Myc tagged) were from Upstate (Charlottesville, VA, USA).

The constitutively active Akt is a Gag fusion protein, with a src

myristoylation signal sequence at its amino terminus. In the case

of dominant negative Akt, a mutation (K179M) results in loss of

affinity for ATP, and consequently a protein that is inactive as a

kinase. SMARTpool Akt siRNA was also from Upstate. Rabbit

polyclonals to total Akt and Ser 473 phosphorylated Akt (p-Akt,

Catalogue no. 9271, specific for Western blotting, and Catalogue

no. 9277, specific for immunocytochemistry) were from Cell

Signalling Technology (Beverly, MA, USA). Anti-c-Myc mono-

clonal antibody (clone 9E10) was from Santa Cruz Biotechno-

logy (Santa Cruz, CA, USA).

Cell culture

NB-4 [an APL cell line carrying the t(15;17) chromosomal

translocation], CEM, Jurkat, and MOLT-4 cells (all T-cell

leukaemia-derived cell lines) were routinely maintained in

Roswell Park Memorial Institute (RPMI) 1640 medium

supplemented with 10% fetal calf serum (complete medium)

at an optimal cell density of 3–8 · 105 cells/ml.

Selection of NB-4 As2O3-resistant cell line(NB-4-ArsRES/AKT)

An As2O3-resistant cell line, hereafter referred to as NB-4-

ArsRES/AKT, was derived from the parental NB-4 cell line

essentially as described by Gianni et al (1998). Briefly, cells

were continuously cultured in the presence of 1 lmol/l As2O3.

While most of the cell population died, rare As2O3-resistant

cells became evident after about 4 months of selection. To

separate the living from the dead cells, the method described

by Thøger-Andersen and Junker (1994) was employed. This

method exploits the specificity of the plant lectin concanavalin

A to bind glucosyl and mannosyl residues that are almost

universally present on the outer surface of mammalian cell

membranes. Before plating in tissue culture plates, the cell

suspension was maintained at 37�C and supplemented with

PI3K/Akt Inhibition and As2O3 in Leukaemia Cells

ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 130, 716–725 717

sterile agarose at the final concentration of 0Æ18%. After

2 weeks, clones were picked with a Pasteur pipette, and

expanded in complete medium. Individual clones were

screened for Ser 473 p-Akt levels by flow cytometry, as

reported elsewhere (Tazzari et al, 2004).

Primary APL blast culture

The APL patient samples were obtained at diagnosis from

peripheral blood after informed consent was given according

to the ethical standards of the institutional guidelines. All

samples had >80% blasts after Ficoll–Hypaque density-gradi-

ent centrifugation, as assessed by flow cytometric analysis with

anti-HLA-DR, CD13, CD33, CD34 antibodies (alone or in

combination). APL blasts (1 · 106/ml) were cultured in

methylcellulose medium (Methocult; Stem Cell Technologies,

Vancouver, Canada) supplemented with human recombinant

growth factors: interleukin (IL)-3 (20 ng/ml), IL-6 (20 ng/ml),

stem cell factor (50 ng/ml). They were treated with for 12 h

with LY294002 (30 lmol/l) and then incubated with As2O3

(1 lmol/l) for 48 h.

cDNA transfection

Transfection of parental NB-4 cells was performed by electro-

poration with a Bio-Rad Gene Pulser apparatus (Neri et al,

2003). Twenty microgram of plasmid DNA (either constitu-

tively active Akt or dominant negative Akt) were mixed with

107 cells in 0Æ5 ml of phosphate-buffered sucrose (272 mmol/l

sucrose and 7 mmol/l Na2HPO4, pH 7Æ4). Cells were electro-

porated with a pulse of 250 V for 18–20 ms. Control cells were

mock-transfected in the same conditions with the empty

vector. Following electroporation, cells were allowed to recover

in 20 ml of culture medium for 48 h before selection with

600 lg/ml of G418. G418-resistant transfected clones were

obtained by limited dilution.

siRNA transfection

Prior to electroporation, CEM, Jurkat and MOLT-4 cells were

washed twice with serum-free Opti-MEM (Gibco BRL, Paisley,

UK) and resusupended to a final concentration of

8 · 106 cells/ml in Opti-MEM. Subsequently, 0Æ5 ml of cell

suspension was mixed with either 0Æ5 nmol of siRNA against

Akt or non-specific control siRNA (as supplied by the

manufacturer) and electroporated in a 0Æ4-cm cuvette using

the Gene Pulser apparatus with a pulse of 260 V and a

capacitance of 1050 lF. Cells were immediately plated in

complete medium and treated with As2O3. After 48 h cells

were analysed by Western blotting for the levels of Akt.

Detection of apoptosis

For detection of apoptosis, cells were stained with Annexin

V-FITC (FITC) (Zhao et al, 2004). Briefly, cells were washed in

phosphate-buffered saline (pH 7Æ4, PBS) and resuspended in

100 ll of binding buffer containing Annexin V-FITC. Cells

were analysed by flow cytometry after addition of propidium

iodide (PI). Annexin V-FITC binds to those cells that express

phosphatidylserine on the outer layer of the cell membrane,

and PI stains the cellular DNA of those cells with a

compromised cell membrane. This enabled live cells (un-

stained with either fluorochrome) to be discriminated from

apoptotic cells (stained only with Annexin V-FITC) and

necrotic cells (stained with both Annexin V and PI) (Vermes

et al, 1995). In some cases, apoptotic cells were detected by the

TUNEL/PI technique, as reported elsewhere (Li et al, 1995;

Gao et al, 2004). Briefly, cells were first fixed at room

temperature with 4% paraformaldeheyde for 30 min and then

permeabilised for 2 min on ice in the presence of 0Æ1% Triton

X-100. Samples were then incubated (60 min at 37�C in the

dark) in the TUNEL reaction mixture containing TdT

(terminal deoxynucleotidyl transferase) and FITC-dUTP,

washed twice in PBS, treated with 100 U/ml RNase A, stained

with PI (50 g/ml), and finally analysed by flow cytometry. TdT

catalyses the polymerisation of the labelled nucleotide to free

3¢-OH DNA ends in a template-independent manner. The

TUNEL reaction preferentially labels DNA strand breaks

generated during apoptosis and enables discrimination

between apoptotic and necrotic cells. Cell cycle analysis was

performed on PI-stained samples as reported elsewhere

(Cappellini et al, 2003).

Intracellular immunostaining for Ser 473 p-Akt and flowcytometric analysis

This was performed essentially as described previously

(Tazzari et al, 2004). Cells (approximately 1 · 105/sample)

were fixed with Reagent 1 of the Intraprep kit, according to

the manufacturer’s instructions (Beckman Coulter, Miami,

FL, USA). Cells were then permeabilised with saponin-

based Reagent 2 and incubated at 4�C for 12 h with a 1:10

final dilution of antibody to Ser 473 p-Akt (Cell Signaling

Technology, catalogue no. 9277, specific for immunocyto-

chemistry). Samples were washed twice with PBS and

pellets were incubated with 5 ll of a FITC-conjugate swine

anti-rabbit IgG (DakoCytomation, Glostrup, Denmark).

Negative controls were run with FITC-conjugated swine

anti-rabbit IgG alone and with FITC-conjugated swine anti-

rabbit IgG plus normal rabbit IgG (from Sigma). All the

samples were analysed by EPICS XL and Cytomics FC 500

flow cytometers (Beckman Coulter) equipped with dedica-

ted software. Histograms were then analysed with EXPO

software.

Protein concentration assay

This was performed according to the instruction of the

manufacturer using the detergent-compatible Bio-Rad Protein

Assay.

G. Tabellini et al

718 ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 130, 716–725

Preparation of cell homogenates and Western blot analysis

Cells were washed twice in PBS, then lysed (107/ml) in

10 mmol/l Tris-HCl, pH 7Æ4, 1 mmol/l MgCl2, 1 mmol/l

EGTA, 1% Triton X-100, 0Æ25 mol/l sucrose, containing the

COMPLETE Protease Inhibitor Cocktail supplemented with

50 mmol/l NaF, 1 mmol/l 2-glycerophosphate, 25 mmol/l

NaPPi. After 15 min on ice, they were homogenised by 10

passages through a 25-gauge needle, and centrifuged in a

microfuge (9300 g) for 10 min at 4�C. An aliquot of the

supernatant was saved for protein assay. Supernatants were

mixed with 4x electrophoresis sample buffer, and then the

protein (80 lg/lane), separated by sodium dodecylsulphate-

polyacrylamide gel electrophoresis (SDS-PAGE), was trans-

ferred to nitrocellulose sheets using a semi-dry blotting

apparatus. Sheets were saturated in PBS containing 5% NGS,

4% BSA, 0Æ25 non-fat dry milk (blocking buffer) for 60 min

at 37�C, then incubated overnight at 4�C in blocking buffer

containing the primary antibody. After four washes in PBS

containing 0Æ1% Tween-20, they were incubated for 30 min

at room temperature with the appropriate peroxidase-conju-

gated secondary antibody, diluted 1:5000 in blocking buffer,

and washed as above. Bands were visualised by the ECL

method.

Statistical evaluation

Data are shown as mean ± SD. For statistical analyses, the

Student’s t-test for unpaired samples at the level of significance

of 0Æ01 was used.

Results

Ser 473 p-Akt levels and As2O3 sensitivity in human acuteleukaemia cell lines

We first analysed by Western blot the expression of total Akt

and the levels of Ser 473 p-Akt in four human acute

leukaemia cell lines. As shown in Fig 1A, NB-4, CEM, Jurkat

and MOLT-4 cells expressed comparable amount of total

Akt. In contrast, a marked Akt phosphorylation on Ser 473

was detected only in CEM, Jurkat and MOLT-4 cells, in

agreement with others (e.g. Xu et al, 2002; Uddin et al,

2004). NB-4 cells were completely negative for Ser 473

p-Akt. The sensitivity of these cells lines to incubation for

48 h with 1 lmol/l As2O3 was analysed next. NB-4, CEM

and MOLT-4 cells were highly sensitive to As2O3 as

indicated by the percentage of Annexin V-positive cells

detected by flow cytometry. On the contrary, Jurkat cells

were resistant to this concentration of As2O3. In some cases,

apoptotic cells were detected by means of the TUNEL/PI

technique with results similar to those obtained with

Annexin V staining (Fig 1B). Overall, our results were in

good agreement with the findings reported elsewhere

(Rojewski et al, 2002; Hu et al, 2003).

Cell cycle analysis

The effect of As2O3 treatment on cell cycle was evaluated by

flow cytometric analysis of PI-stained samples. As2O3

increased the percentage of NB-4 cells in the G2/M phase of

the cell cycle along with an increase in sub-G1 (apoptotic

cells), in agreement with others (e.g. Gao et al, 2004).

Consistent with the lack of Akt phosphorylation, PI3K

A

B

Fig 1. Akt expression, phosphorylation levels, and As2O3 sensitivity of

acute human leukaemia cell lines. (A) Western blot analysis for total

Akt and Ser 473 p-Akt. Protein (80 lg/lane) was separated by SDS-

PAGE and blotted to nitrocellulose sheets. Immunoreactive bands were

visualised by the ECL technique. For detection of p-Akt, antibody no.

9271 was employed. Immunostaining with a monoclonal antibody to

b-tubulin confirmed equal loading. Blots are representative of three

separate experiments. (B) Flow cytometric analysis of As2O3-induced

apoptosis. Cells were treated with 1 lmol/l As2O3 for 48 h. The per-

centage of apoptotic cells was evaluated by Annexin V-FITC staining or

TUNEL/PI technique. Untreated cells were negative for TUNEL reac-

tion (not shown). A representative of three separate experiments is

shown.



inhibitors did not affect the NB-4 cell cycle in a substantial

way, and they did not also increase the number of apoptotic

cells in association with As2O3 (Fig 2A). In contrast, As2O3 did

not increase the percentage of MOLT-4 cells in the G2/M

phase of the cell cycle, but rather it directly induced apoptosis

(Fig 2B). PI3K inhibitors increased As2O3-dependent apopto-

sis of MOLT-4 cells. Similar results were obtained with CEM

cells (not shown). As Jurkat cells were not sensitive to As2O3,

we did not investigate their cell cycle.

Modulation of the PI3K/Akt pathway by genetictechniques affects As2O3 sensitivity of NB-4 cells

Very recent results have highlighted that treatment with the

PI3K/Akt pathway inhibitor LY294002 markedly increased the

sensitivity of NB-4 cells to As2O3 (Ramos et al, 2005). These

results appeared surprising in light of the finding that our NB-

4 cells did not display Akt phosphorylation. Therefore, we

pretreated NB-4 cells with either LY294002 (30 lmol/l) or

wortmannin (0Æ5 lmol/l) for 12 h, then incubated them with

1 lmol/l As2O3 for 48 h. As shown in Fig 3A, the inhibitors,

used alone or in combination with As2O3, did not significantly

increase the percentage of apoptotic NB-4 cells in comparison

with controls.

We then employed a genetic approach for upregulating Akt

activity in NB-4 cells to determine whether this would result in

a decreased As2O3 sensitivity. NB-4 cells were stably transfected

with either a constitutively active Akt or a dominant negative

Akt and clones were selected. As presented in Fig 3B, two clones

(18E and 21D) expressed almost equal levels of constitutively

active Akt, as demonstrated by Western blot analysis with an

A

B

C

Fig 3. Modulation of the PI3K/Akt pathway affects As2O3 sensitivity

of NB-4 cells. (A) Cells were incubated for 12 h with either LY294002

(30 lmol/l) or wortmannin (0Æ5 lmol/l), then exposed to 1 lmol/l

As2O3 for 48 h. Samples were analysed for apoptotic cells using

Annexin V-FITC staining. Results are the mean of three different

experiments ± SD. (B) Western blot analysis for the expression of

constitutively active Akt or dominant negative Akt in stably transfected

NB-4 cell clones. Protein (80 lg/lane) was separated by SDS-PAGE

and blotted to nitrocellulose sheets. Blots were probed with an anti-

body to the c-Myc tag of Akt cDNAs, hence parental (WT ¼ wild

type) NB-4 cells were negative. Immunoreactive bands were visualised

by the ECL technique. (C) Sensitivity of NB-4 cell clones to As2O3.

Samples were incubated for 48 h in the presence of 1 lmol/l As2O3,

then the percentage of apoptotic cells was evaluated by flow cytometry

using Annexin V-FITC staining. Results are the mean of three different

experiments ±SD. The asterisks indicate significant differences

(P < 0Æ01) in comparison with parental (WT) cells. MT, mock-trans-

fected cells.

Fig 2. Cell cycle analysis of cells treated with As2O3 and/or PI3K

inhibitors. (A) NB-4 cells; (B) MOLT-4 cells. Cells were incubated for

12 h with either LY294002 (30 lmol/l) or wortmannin (0Æ5 lmol/l),

then exposed to 1 lmol/l As2O3 for 48 h. Flow cytometric analysis was

performed on PI-stained samples. A representative of three separate

experiments is shown. APO: apoptotic cells. Asterisks indicate signi-

ficant differences (P < 0Æ01) in comparison with untreated cells.

G. Tabellini et al


antibody to the c-Myc tag. Clones 11A and 13Y expressed a

comparable amount of dominant negative Akt.

When the As2O3 sensitivity of these clones was analysed, it

became evident that the two clones that overexpressed

constitutively active Akt were significantly less sensitive to

1 lmol/l As2O3 than wild type NB-4 cells or mock transfected

(i.e. transfected with the empty vector) cells. In contrast, cell

clones overexpressing a dominant negative form of Akt were as

sensitive to As2O3 as wild type cells (Fig 3C).

Selection of an As2O3 resistant NB-4 cell subline with highlevels of Ser 473 p-Akt

Previous results have indicated that it is possible to select NB-4

cell sublines that are resistant to As2O3 (e.g. Gianni et al,

1998). Moreover, recent findings have highlighted that an

As2O3-resistant NB-4 cell subline exhibited higher levels of

ERK1/2 activity and that downregulation of this survival

pathway markedly decreased As2O3 resistance (Lunghi et al,

2005). Taking also into account that exposure to apoptotic

stimuli could result in the selection of a HL60 cell subline with

an active PI3K/Akt signalling pathway (Neri et al, 2003), we

decided to investigate whether As2O3 resistance of NB-4 cell

sublines might somehow be related to upregulation of Akt

signalling. As2O3-resistant NB-4 cells were selected from

parental NB-4 cells treated with 1 lmol/l As2O3 over a

4-month period. Cell clones were then selected and screened

by flow cytometry (Tazzari et al, 2004) for high levels of Ser

473 p-Akt.

A cell subline with high levels of Ser 473 p-Akt was identified

(NB-4-ArsRES/AKT) and further analysed by Western blotting

(Fig 4A). The amount of total Akt detected in NB-4-ArsRES/

AKT was similar to that of parental NB-4 cells. However, also by

Western blotting, NB-4-ArsRES/AKT cells displayed a very high

level of Ser 473 p-Akt. Ser 473 p-Akt levels could be effectively

downregulated by incubation with two pharmacological

inhibitors of the PI3K/Akt pathway, LY294002 and wortman-

nin. As a control, we also analysed an As2O3-resistant clone,

referred to as NB-4-ArsRES, which did not display high levels of

Ser 473 p-Akt (Fig 4A) by Western blot.

The NB-4-ArsRES/AKT cells were much less sensitive to

1 lmol/l As2O3-induced apoptosis than parental cells (Fig 4B).

Treatment with either LY294002 or wortmannin significantly

increased the number of NB-4-ArsRES/AKT cells that underwent

apoptosis in response to As2O3 incubation, whereas LY294002

or wortmannin per se did not significantly increase the

percentage of apoptotic cells. In contrast, As2O3-induced

apoptosis of NB-4-ArsRES was not influenced at all by the

presence of LY294002 (Fig 4C).

PI3K/Akt inhibition enhances As2O3 sensitivity in APLprimary blasts

We next studied the effect of LY294002 on As2O3-dependent

apoptosis of primary APL blasts. Due to the limitations caused

by the amount of the samples, we decided to analyse the levels

of Ser 473 p-Akt of APL blasts prior to and after PI3K/Akt

inhibition by flow cytometry (Tazzari et al, 2004). Indeed, this

technique requires much fewer cells than Western blotting and

gives the same results. As controls we employed CEM cells,

CEM cells treated with LY294002, and NB-4 cells. LY294002

caused a dramatic fall in the levels of Ser 473 p-Akt, as revealed

by Western blotting (data not shown, but see Uddin et al,

2004). Flow cytometric analysis confirmed the positivity of

A

B

Fig 4. An NB-4 cell clone with high levels of Ser 473 –Akt (NB-4-

ARSRES/AKT) is resistant to As2O3-induced apoptosis. (A) Western blot

analysis for total Akt and Ser 473 p-Akt. Cells were incubated for 12 h

with either LY294002 (30 lmol/l) or wortmannin (0Æ5 lmol/l), then

lysed for SDS-PAGE and Western blot analysis. Immunostaining with

a monoclonal antibody to b-tubulin confirmed equal loading. Blots are

representative of three separate experiments. (B and C) Flow cyto-

metric analysis. NB-4-ARSRES/AKT (B) or NB-4-ARSRES (C) cells were

first incubated for 12 h with solvent carrier (dimethylsulphoxide) or

PI3K/Akt inhibitors (LY294002, 30 lmol/l; wortmannin, 0Æ5 lmol/

l ¼ WORT), then exposed to 1 lmol/l As2O3 for 48 h and analysed

by flow cytometry. The percentage of apoptotic cells was evaluated

by Annexin V-FITC staining (B) or TUNEL/PI technique (B, C).

Untreated cells were negative for TUNEL reaction (not shown).

A representative of three separate experiments is shown.



CEM cells for Ser 473 p-Akt and its decrease in LY294002-

treated samples (Fig 5A). NB-4 cells were negative for Ser 473

p-Akt, in agreement with Western blot data (see Fig 1). Of the

four APL patients, two were positive and two negative for Ser

473 p-Akt. In the two positive cases, incubation with LY294002

(12 h at 30 lmol/l) resulted in downregulation of the quantity

of Ser 473 p-Akt.

We next studied the apoptotic effect of the combined

treatment (LY294002 + As2O3) in APL blasts (Fig 5B). When

employed alone, LY294002 did not increase apoptosis in a

significant manner. The two samples with activated Akt were

less sensitive to As2O3 (either 1 or 2Æ5 lmol/l) than those

without activation. In p-Akt positive samples, even if the

As2O3 concentration was raised to 2Æ5 lmol/l, the percentage

of apoptotic cells was inferior to that detected in p-Akt

negative samples incubated with 1 lmol/l As2O3. The com-

bined treatment (1 lmol/l As2O3 + LY294002) resulted in a

dramatic increase of apoptotic cells only in samples with high

levels of activated Akt.

Abrogation of Akt expression by siRNA sensitises acuteT-leukaemia cell lines to As2O3

Recently, siRNAs have also been shown to achieve a high

degree of specificity with low toxicity in mammalian cells

acting through a degradative chain reaction catalysed by the

activation of a cellular RNA-dependent RNA polymerase

(Caplen et al, 2001; Elbashir et al, 2001). While much work

remains to optimise delivery and maintain specificity, the

therapeutic advantages of siRNAs for treatment of cancers, in

which genes are upregulated or misappropriately expressed,

show great promise (Ryther et al, 2005).

We therefore, decided to selectively knock-down Akt in

CEM, Jurkat and MOLT-4 cells by means of siRNA for

increasing sensitivity to As2O3. Transfection of Akt siRNA, but

not of the non-specific control siRNA, led to the decrease of

Akt in these cell lines without affecting the unrelated protein,

tubulin (Fig 6A). A significant increase in the number of

apoptotic cells in response to 1 lmol/l As2O3 treatment for

48 h was seen in samples in which Akt expression was

abrogated by siRNA (Fig 6B).

Discussion

The results presented in this work indicate that the generation

of apoptosis by 1 lmol/l As2O3 is potentiated by down-

modulation of the PI3K/Akt pathway in APL and T-leukaemia

cells. As2O3 also affected the cell cycle of NB-4 cells in that it

increased the number of G2/M phase cells. Therefore, it could

be suggested that apoptotic NB-4 cells derive from cells

blocked in G2/M, in agreement with the findings of others

(Gao et al, 2004). In contrast, As2O3 did not alter the cell cycle

of MOLT-4 or CEM cells but rather it directly induced

apoptosis. Conceivably, these differences are dependent on the

specific cell type.

As to NB-4 cells, a recent report showed that treatment with

LY294002 resulted in increased sensitivity to 1 lmol/l As2O3

(Ramos et al, 2005). However, these authors did not check, by

Western blot, whether their NB-4 cells had elevated levels of

Ser 473 p-Akt and whether or not there was a decrease in Akt

phosphorylation in response to LY294002. The NB-4 cells we

used for this work completely lacked Ser 473 p-Akt and,

consistently, were not further sensitised to 1 lmol/l As2O3 by

treatment with either LY294002 or wortmannin. Nevertheless,

they could be induced to become resistant to As2O3 by

overexpression of a constitutively active Akt cDNA, whereas a

dominant negative Akt cDNA was without effect. Conceivably,

Fig 5. Inhibition of PI3K/Akt sensitises APL blasts to As2O3. (A) flow

cytometric analysis of Ser 473 p-Akt in CEM and NB-4 cells, and APL

blasts. Cells were incubated for 12 h in the presence of LY294002

(30 lmol/l), then immunostained with antibody to Ser 473-Akt spe-

cific for immunocytochemistry. Controls were run with FITC-conju-

gated swine anti-rabbit IgG alone and with FITC-conjugated swine

anti-rabbit IgG plus normal rabbit IgG. (B) Flow cytometric analysis of

As2O3-induced apoptosis. Cells were incubated for 12 h with either

LY294002 (30 lmol/l) or an equivalent volume of solvent carrier

(dimethylsulphoxide), then exposed for 48 h to As2O3. The percentage

of apoptotic cells was then evaluated by Annexin V-FITC staining.

Results are the mean ± SD of a single experiment performed in trip-

licate.

G. Tabellini et al


the apparent discrepancy between their findings and ours

could be explained by the fact the NB-4 cells employed by

Ramos et al (2005) had elevated levels of active Akt.

Our results also indicate that, upon prolonged exposure to

As2O3, it was possible to isolate a cell subline, referred to as

NB-4-ArsRES/AKT, which displayed elevated levels of Ser 473

p-Akt and was resistant to 1 lmol/l As2O3. Previous reports

have indicated that As2O3 down-modulated Ser 473 p-Akt

levels of U937 cells (Choi et al, 2002; Ramos et al, 2005).

However, our results indicate that it is also possible that, in

response to As2O3, there is an upregulation of the PI3K/Akt

survival pathway that causes resistance to As2O3. In this

connection, it should be emphasised here that Akt can be

activated by many forms of cellular stress, including heat

shock, ultraviolet light, ischaemia, hypoxia and oxidative stress

(reviewed in West et al, 2002, and references therein). Stress-

induced Akt upregulation is likely to be viewed as a compen-

satory protective mechanism that cells activate to escape death.

Interestingly, the production of reactive oxygen species may be

stimulated by As2O3 in human myeloid leukaemia cells

(Perkins et al, 2000), and this might represent a sufficient

stimulus for the activation of Akt. We feel that the results

obtained with the NB-4-ArsRES/AKT cell subline might have

important consequences at the clinical level, as they might help

explaining the occurrence of resistance to As2O3. Indeed, early

relapses from As2O3 treatment within a few months were not

infrequently seen in APL patients (Huan et al, 2000). There-

fore, it would be interesting to investigate whether or not Akt

activation might be implicated in As2O3 also in APL patients.

In this connection, it is worthwhile to note that APL blasts

with activated Akt were less sensitive to As2O3 than those with

no Akt activation. Although the number of investigated

patients was low, we feel that these findings are suggestive

for a role of PI3K/Akt also in the resistance of APL patients to

As2O3 treatment in vivo. It might be hypothesised, for example,

that patients who are resistant to As2O3 have extremely

elevated levels of Ser 473 p-Akt. Indeed, the phosphorylation

levels of this residue have been demonstrated to display a great

variability among patients with AML (Xu et al, 2003).

However, it is important to emphasise that our findings seem

to indicate that activation of the PI3K/Akt pathway cannot be

considered per se the only mechanism that leads to As2O3

resistance in NB-4 cells and T-leukaemia cell lines. Indeed, an

As2O3-resistant clone (NB-4-ArsRES) was isolated that did not

display elevated levels of Akt. Moreover, CEM, Jurkat and

MOLT-4 cells have elevated basal levels of Ser 473 p-Akt, due to

the lack of the lipid phosphatase PTEN which downregulates

the levels of 3¢ phosphorylated inositol lipids (Uddin et al,

2004). While CEM and MOLT-4 were sensitive to 1 lmol/l

As2O3, Jurkat cells were resistant. However, Akt knock-down by

siRNA increased 1 lmol/l As2O3 sensitivity of all of these three

cell lines. Therefore, it is conceivable that other signalling

networks also mediate As2O3 resistance of APL cells, for

example the Erk 1/2 survival pathway (Lunghi et al, 2005).

In conclusion, our results strengthen the hypothesis that the

PI3K/Akt pathway may be one of the factors important for

resistance to As2O3-induced apoptosis in a variety of human

acute leukaemia cell lines. Moreover, our findings indicated

that down-modulation of this survival pathway could increase

APL blast sensitivity to As2O3. Therefore, it might be that

future innovative therapeutic strategies for acute leukaemias

A

B

Fig 6. Abrogation of Akt expression sensitises acute T-leukaemia cell

lines to As2O3. (A) Western blot analysis. Cells were transfected with

the indicated siRNA for 48 h. Subsequently, cells were harvested for

Western blot analysis, which was performed as indicated in legend to

Fig 1. (B) Samples in which Akt had been knocked-down by siRNA

transfection, were incubated for 48 h in the presence of 1 lmol/l As2O3

for 48 h, then the percentage of apoptotic cells was evaluated by flow

cytometry using Annexin V-FITC staining. Results are the mean of

three different experiments ±SD. Asterisks indicate significant differ-

ences (P < 0Æ01) in comparison with untreated cells.



will consist of inhibition of the PI3K/Akt pathway in addition

to As2O3. PI3K/Akt inhibition might be achieved not only

through the use of selective pharmacological inhibitors, but

also by means of siRNA, which is rapidly establishing itself as a

very promising technology for specific gene silencing also in

haematological oncology (Woessmann et al, 2003).

In this regard, it is worth emphasising that the first phase I

clinical trial involving a PI3K/Akt inhibitor, perifosine (Kon-

dapaka et al, 2003), was recently performed on patients with

incurable solid malignancies. These results suggested perifosine

activity in sarcoma and perhaps renal cell carcinoma, thus

justifying additional investigation of this agent in a phase II

sarcoma trial (Van Ummersen et al, 2005). Thus, perifosine

might also be employed for the treatment of patients with

haematological malignancies in the future.

Acknowledgements

This work was supported by grants from: AIRC, Italian MIUR

Cofin 2003 and FIRB 2001, Selected Topics Research Fund

from Bologna University, ‘Hairshow A.I.L.’, Fondazione del

Monte di Bologna e Ravenna.

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