Haematologica 2003 - Supplements

157
The Nitrogen-Containing Bisphosphonate, Zoledronic
Acid Regulates RANKL Expression in Human
Osteoblast-Like Cells, by Activating TNF-alpha
Converting Enzyme (TACE)
Beiqing Pan, Amanda N. Farrugia, Luen Bik To, and
Andrew C.W. Zannettino
Institute of Medical and Veterinary Science
Bisphosphonates (BPs) exhibit high affinity for hydroxyapatite
mineral in bone and are used extensively to treat the osteolytic bone
disease and hypercalcaemia seen in most patients with myeloma.
The BP-mediated inhibition of bone resorption has been attributed
mainly to their effects on the proliferation and apoptosis of bone–
resorbing osteoclasts (OCs). In the present study, we examined the
effect of the nitrogen containing BP, zoledronic acid (ZOL), on the
expression of RANKL and OPG, critical factors in the regulation of
OC formation and activation, in primary human osteoblast (OB)-like
cells. Our studies show that ZOL, whilst not significantly affecting
RANKL or OPG gene expression, markedly increased OPG protein
secretion and reduced membrane RANKL protein expression. The
reduction in membrane RANKL expression was preceded by a
marked increase in the expression of the metalloprotease-disintegrin
TNF-alpha converting enzyme (TACE) in OB-like cells. In addition,
the decreased membrane expression of RANKL could be partially
reversed by a TACE inhibitor, TAPI-2. ZOL not only caused an
increase in TACE expression, but also appeared to mediate a redistribution
of the TACE protein from a nuclear to perinuclear
compartment in OB-like cells, raising the possibility that TACE may
function intracellularly to cleave RANKL protein prior to its
expression at the cell surface. Therefore, our studies indicate that
ZOL, in addition to its direct effects on mature OCs, may inhibit the
recruitment of pre-OCs by decreasing the level of membraneassociated
RANKL expression in OB-like cells.
158
Zoledronic acid inhibits the proliferation and induces
apoptosis of bone marrow stromal cells in multiple
myeloma
Alessandro Corso, Eleonora Ferretti, Mariangela Maiocchi,
Patrizia Zappasodi, Silvia Mangiacavalli, Angela Lorenzi,
Chiara Rusconi, Marzia Varettoni, Monia Lunghi, Mario
Lazzarino
Division of Hematology, IRCCS Policlinico San Matteo, University
of Pavia, Pavia, Italy
Bisphosphonates are commonly used to prevent bone resorption
in multiple myeloma. In vitro studies have demonstrated their
capacity of inhibiting the proliferation of a variety of human
tumor cells lines as well as decreasing their viability and inducing
apoptosis. On the contrary, few evidences are reported on their
effects on stromal cells, in particular in myeloma patients. To
explore the anti-tumor activity of bisphosphonates against clonal
plasma cells and to test their impact on bone marrow
microenvironment, we investigated the cytostatic and apoptotic
effects of Zoledronate on the human myeloma cell line RPMI
8226 and bone marrow stromal cells (BMSCs) isolated from
seven patients with active multiple myeloma.
Bone marrow collected from MM patients was diluted with an equal
volume of Dulbecco's Phosphate Buffered Saline (PBS), separated on
Ficoll-Hypaque (density = 1077). Mononuclear cells were then
incubated in culture flask T75 in medium "MyelocultTM" with added
100 U/ml Penicillin, 100 U/ml Streptomycin and Hydrocortison at
37°C in 5% CO2. The cultures were weekly fed by replacing 50% of
the supernatant with fresh culture medium, until a confluent adherent
cell monolayer was obtained (2-3 weeks). The anti-proliferative
effect of Zoledronate was evaluated at escalating concentrations of
the drug (10µM-500µM) for 72h, using the MTT assay on BMSCs
and on 8226 cell line. To determine whether the reduction in cell
proliferation observed with zoledronic acid treatment was sustained
by apoptotic death, 8226 cells and BMSCs were analyzed by flow
cytometric detection of fuorescein labelled Annexin V.
Our results show that zoledronic acid induces a dose-dependent
inhibition of proliferation of BMSCs and of 8226 cells; furthermore,
the addition of 5ng/ml rhIL-6 to myeloma cells does not revert the
antiproliferative effect of Zoledronate. Apoptosis induced by
Zoledronate in human myeloma cell line was daily evaluated at
concentration of 10-4 M or 5x10-4 M for five days. The percentage
of apoptotic cells was time- and dose-dependent; in particular a
stronger effect was observed at the concentrationof 500µM. Given
the ability of Zoledronate to significantly reduce cell proliferation of
BMSCs, we also performed flow cytometric analysis of apoptotic
cells. After 3 days of treatment with bisphosphonate, approximately
7% of the adherent cells were found to be positive for Annexin V
binding at 10-5 M of Zoledronate, compared to 3-4% in untreated
controls; the percentage of apoptotic cells increased significantly at a
concentration of 10-4 M.
In conclusion, these results demonstrate an antiproliferative and
pro-apoptotic activity in vitro of zoledronic acid on bone marrow
stromal cells and on myeloma cell lines, and support a possible
anti-tumor effect in vivo of this drug.
159
OPG can Protect Human Myeloma Cells Against TRAILinduced
Apoptosis; A Role for OPG as a Paracrine
Survival Factor in Multiple Myeloma?
Claire M. Shipman & Peter I. Croucher
Nuffield Dept. of Orthopaedic Surgery, University of OxfordUK
Multiple myeloma is a haematological malignancy in which the tumour
cells grow preferentially within the bone marrow microenvironment.
Within this local environment myeloma cells can interact with a range
of different cell types including osteoblasts, bone marrow stromal cells
(BMSCs) and osteoclasts. These interactions are critical both for
tumour growth and the development of the associated bone disease.
Osteoprotegerin (OPG) is a member of the tumour necrosis factor
(TNF) receptor superfamily. OPG binds to the ligand for receptor
activator of nuclear factor B (RANKL), and prevents the interaction
between RANKL and RANK, thus inhibiting osteoclast formation and
bone resorption. The RANKL/OPG system appears to play an
important role in the development of myeloma bone disease. However,
it is unclear whether OPG may be able to bind to other TNF family
members, such as TNF-related apoptosis-inducing ligand (TRAIL),
and, by inhibiting their activity, function as a survival factor for
myeloma cells. The aim of the present study was to determine whether
OPG released from osteoblasts could protect human myeloma cells
against TRAIL-induced apoptosis.
Apoptotic cells were identified by characteristic changes in nuclear
morphology and by a fluorescence in situ nick translation assay.
Apoptosis was measured in mononuclear cells isolated from the bone
marrow of patients with multiple myeloma or cocultures of myeloma
cells and osteoblasts cells by two colour flow cytometry. Myeloma
cells were identified by Ig light chain expression and apoptosis by a
fluorescence in situ nick translation assay. Recombinant OPG
significantly protected RPMI 8226 and NCI H929 myeloma cells
against TRAIL-induced apoptosis in a dose-dependent manner (p<
0.001). Recombinant OPG was also shown to inhibit TRAIL-induced
apoptosis of primary human myeloma cells isolated from the bone
marrow of a patient with multiple myeloma (p