Leukemia Research 28 (2004) 387–397
Peroxisome proliferator-activated receptor
ligands induce growth
inhibition and apoptosis of human B lymphocytic leukemia
Chuanbing Zang a,1, Hongyu Liu a,1, Maximilian G. Posch a, Maries Waechter a,
Margit Facklam a, Martin H. Fenner a, Martin Ruthardt b, Kurt Possinger a,
H. Phillip Koeffler c, Elena Elstner a,∗
a Division of Oncology/Hematology, School of Medicine (Charité), Humboldt University, Schumannstr. 20/21, 10117 Berlin, Germany
b Laboratory for Experimental Hematology, Division of Hematology, School of Medicine, University of Frankfurt,
Theodor Stern Kai 7, 60590 Frankfurt/Main, Germany
c Division of Hematology/Oncology, Cedars Sinai Medical Center, UCLA, Los Angeles, CA, USA
Received 6 February 2003; accepted 31 July 2003
Abstract
This study examined the expression and structural intactness of peroxisome proliferator-activated receptor
(PPAR
) in human acute
lymphocytic leukemia (ALL) cells and determined the effect of PPAR
ligands on growth and apoptosis of these cells. We noted that all
lymphocytic leukemia cell lines expressed PPAR
and no PPAR
mutations were found in these cell lines as indicated by SSCP analysis.
Effect of the PPAR
ligands on the proliferation, differentiation and apoptosis of B type ALL cells was further examined. Treatment of these
cells with the PPAR
ligands Pioglitazone (PGZ) and 15-deoxy-delta (12,14)-prostaglandin J2 (15d-PGJ2) resulted in growth inhibition in
a dose-dependent manner which was associated with a G1 to S cell cycle arrest. However, this effect appeared to be PPAR
-independent
since several PPAR
antagonists could not reverse this effect. No differentiation was induced by this treatment. Four out of five cell lines
underwent apoptosis after culture with the PPAR
ligands. This effect was partially caspase-dependent because a pan-caspase inhibitor
partially reversed this effect. In conclusion, our results suggest that PPAR
ligands may offer a new therapeutic approach to aid in the
treatment of ALL.
© 2003 Elsevier Ltd. All rights reserved.
Keywords: Acute lymphocytic leukemia; Peroxisome proliferator-activated receptor
; Pioglitazone; Proliferation; Apoptosis
1. Introduction
Acute lymphocytic leukemia (ALL) is a heterogeneous
group of diseases in which the malignant clone arises from
the uncontrolled proliferation of lymphocytic progenitors in
the bone marrow or lymphatic system. Although 80–90%
of ALL patients can reach complete remission with cur-
Abbreviations: ALL, acute lymphocytic leukemia; LFS, leukemia-free
survival; BADGE, bisphenol A diglycidyl ether; CDKs, cyclin depen-
dent kinases; CDKIs, cyclin dependent kinase inhibitors; 15d-PGJ2,
15-deoxy-delta (12,14)-prostaglandin J2; NHRs, nuclear hormone recep-
tors; (Ph+) ALL, Philadelphia chromosome-positive acute lymphocytic
leukemia; TGZ, Troglitazone; PGZ, Pioglitazone; PPAR
, peroxisome
proliferator-activated receptor gamma; TZD, Thiazolidinedione; SSCP,
single strand conformational polymorphism; CT, threshold cycle; TUNEL,
terminal deoxynucleotidyl-transferase-mediated UTP nick end labelling
∗ Corresponding author. Tel.: +49-30-450513243;
fax: +49-30-450513950.
E-mail address: elena.elstner@charite.de (E. Elstner).
1 The two authors contributed equally to this work.
rent treatment protocols, many individuals eventually re-
lapse, and only about 35% of individuals have a long-term
leukemia-free survival (LFS) [1]. The outcome in adult ALL
is not uniform and varies considerably by ALL subtypes. In
adult T-ALL and mature B-ALL, the LFS rates can reach
50%; whereas in Philadelphia chromosome-positive (Ph+)
ALL, the outcome is very poor with a LFS of less than 10%.
In childhood ALL, about 70% of patients can be cured by
the use of combination chemotherapy. However, even with
a modern treatment protocol, about one-third of patients re-
lapse with a disease that was more resistant to therapy re-
sulting in a cure rate less than 50% after the second round
of induction therapy. Therefore, development of effective,
novel therapies for ALL patients with refractory or relapsed
ALL is needed.
Peroxisome proliferator-activated receptors (PPARs) be-
long to the family of nuclear hormone receptors (NHRs) that
include estrogen, thyroid hormone receptors, retinoic acid
and Vitamin D3 receptors as well as retinoid X receptors
0145-2126/$ – see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.leukres.2003.07.005

388 C. Zang et al. / Leukemia Research 28 (2004) 387–397
(RXRs). These receptors are ligand-activated transcription
factors which directly regulate the transcription of target
genes. To date, three subtypes of PPAR (, / and
)
have been identified which exhibit distinct tissue distribu-
tion and are associated with selective ligands. PPAR
is one
of the best characterized NHRs and is directly activated not
only by a naturally occurring arachidonic acid metabolite
15-deoxy-delta (12,14)-prostaglandin J2 (15d-PGJ2) [2,3],
but also by synthetic ligands such as those belonging to the
antidiabetic thiazolidinedione (TZD) class of compounds
[4,5] and certain nonsteroidal anti-inflammatory drugs [5].
Upon ligand binding, PPAR
forms a heterodimer with RXR
which binds to specific sequences usually in the 5′ region of
target genes, known as a peroxisome proliferator response
element (PPRE), and enhances the transcription of these
genes.
PPAR
was initially noted to be highly expressed in
adipose tissue and was found to have a regulatory function
in adipocyte differentiation, insulin sensitization and lipid
metabolism [6–9]. Later studies, however, have demon-
strated that PPAR
is expressed in a variety of types of
cancer cells and has crucial roles in suppressing cell growth
[10], promoting differentiation [11,12] and/or apoptosis
[13–17]. Activation of PPAR
by either TZDs such as
Troglitazone (TGZ), Pioglitazone (PGZ), Ciglitazone or
15d-PGJ2 leads to either terminal differentiation, inhibi-
tion of cell growth and/or apoptosis of tumor cells in a
variety of cancers such as liposarcomas [18,19], prostate
[20,21], colon [22–25], breast [26–28], gastric [13,29], lung
[14,15] and pancreatic [30,31] cancers, as well as myeloid
leukemias [32]. These data suggest that PPAR
agonists
may represent a promising, novel therapeutic approach for
certain human malignancies.
No published data are available about the role of PPAR

in human ALL. In order to understand the role of PPAR
in
ALL and the possible clinical application of PPAR
ligands
in the treatment of ALL, we investigated the expression of
PPAR
in human ALL cell lines and the effects of PPAR

ligands PGZ and 15d-PGJ2 on the cell proliferation and
apoptosis of these transformed cells. Our results show that
PPAR
is expressed in human ALL cells and treatment of
Table 1
Characteristics of cell lines used in this study
Cell lines Cell type Age/sex of patient Specimen site Disease status PPAR
mutations Ph
BV173 BCPa 45/M PB CML, lymphoid blast crisis wt +
CCRF-CEM T 3/F PB ALL, 2nd relapse wt −
IM-9 BLC F BM Myeloma wt −
Jurkat T 14/M PB ALL, 1st relapse wt −
Molt-3 T 19/M PB ALL, relapse wt −
Molt-4 T 19/M PB ALL, relapse wt −
Nalm-6 BCP 19/M PB ALL, relapse wt −
SD-1 BLC F PB ALL, diagnosis wt +
Sup-B15 BCP 9/M BM ALL, 2nd relapse wt +
a Abbreviation: Ph, Philadelphia chromosome; BCP, B-cell progenitors; T, T-cells; BLC, B lymphoblastoid cells; PB, peripheral blood; BM, Bone
marrow; Ph, Philadelphia chromosome; wt, wild type.
ALL cells with PPAR
ligands leads to cell cycle arrest
and/or apoptosis in most of ALL cell lines.
2. Materials and methods
2.1. Materials
The PPAR
ligand PGZ was kindly provided by TAKEDA
Chemical Industries Ltd. (Osaka, Japan) and 15d-PGJ2 was
purchased from Calbiochem-Novabiochem GmBH (Bad
Soden, Germany). Both were dissolved in ethanol at 10−2 M
as stock solutions. The PPAR
agonist GW7845 and antag-
onist GW9662 were gifts from GlaxoSmithKline (Hertford-
shire, UK) and dissolved in DMSO at 10−2 M as a stock
solution. The PPAR
antagonist T0070907 was from Tularik
Inc. (San Francisco, USA), BADGE from Sigma–Aldrich
(Taufkirchen, Germany). All stock solutions were stored
at −70 ◦C and were further diluted to appropriate concen-
trations with medium before use. Ex vivo leukemia blasts
were isolated from blood of ALL patients as usual.
2.2. Cell culture
All cell lines used in this study were purchased from
American Type Culture Collection (Rockville, MD) and
maintained in RPMI-1640 medium supplemented with 10%
fetal calf serum. Cells in logarithmic growth phase were used
for further experiments. The cell lines used in this study are
summarized in Table 1. BV173 is a B-cell precursor cell
line which derived from a chronic myeloid leukemia patient
in blast crisis [33]. IM-9 was derived from the blood of a
patient with multiple myeloma. This cell line later has been
shown not to be a myeloma cell line but a B lymphoblastoid
cell line [34]. All other cell lines are ALL-derived.
2.3. Single strand conformational polymorphism (SSCP)
analysis
DNA isolation and purification were performed by re-
ferring to the user manual of Qiagen DNA isolation Mini