Download Expression of the RET Receptor Tyrosine Kinase and GDNFR

Expression of the RET Receptor Tyrosine Kinase and GDNFR- a in Normal
and Leukemic Human Hematopoietic Cells and Stromal Cells of the Bone
Marrow Microenvironment
By Valter Gattei, Angela Celetti, Aniello Cerrato, Massimo Degan, Angela De Iuliis, Francesca Maria Rossi,
Gennaro Chiappetta, Claudia Consales, Salvatore Improta, Vittorina Zagonel, Donatella Aldinucci, Valter Agosti,
Massimo Santoro, Giancarlo Vecchio, Antonio Pinto, and Michele Grieco
The RET proto-oncogene product is a receptor tyrosine kinase representing the signal-transducing molecule of a
multisubunit surface receptor complex for the glial cell linederived neurotrophic factor (GDNF), in which a novel glycosyl-phosphatidylinositol (PI)-linked protein (termed GDNFRa) acts as the ligand-binding component. We have analyzed
expression of RET and GDNFR-a in purified normal hematolymphopoietic cells, leukemia/lymphoma cell lines, and 154
primary samples from patients with hematopoietic malignancies encompassing different lineages and differentiation
stages. Relatively low amounts of RET mRNA were found in
early CD34" hematopoietic progenitors, but RET transcripts
appeared to increase after myelomonocytic maturation. No
expression of RET was found in peripheral blood and tissue
B and T lymphocytes. Analysis of human myelomonocytic
cell lines was overall consistent with results obtained on
purified normal cells. Accordingly, RET expression was
mainly confined to acute myeloid leukemias (AMLs) displaying either monocytic (French-American-British M4 and
M5) or intermediate-mature myeloid (M2 and M3) phenotypes, being less frequently detected in early myeloid (M0
and M1) AMLs. In contrast, RET mRNA was sporadically detected in B-cell tumors, whereas, among T-cell malignancies,
RET transcripts were mainly detected in cells of postthymic
and mature T-cell phenotype. RET broad detection in primary tumors was not paralleled by the mutual expression
of GDNFR-a, which was detected only in 2 isolated primary
samples and in 3 leukemia/lymphoma cell lines. However,
GDNFR-a transcripts, in the absence of RET mRNA, were
found in normal bone marrow stromal cells (BMSC), in BM
fibroblasts, and in two osteoblast cell lines previously described to support normal hematopoiesis. In the presence
of GDNF-receptors derived from BMSC by PI-specific phospholipase C cleavage, GDNF efficiently bound RET-expressing AML blasts and was functionally active by reducing their
clonogenic growth and triggering the monocytic maturation
of leukemic cells.
q 1997 by The American Society of Hematology.
H
subunit receptor complex for the glial cell line-derived neurotrophic factor (GDNF),15,16 a potent neutrophic factor
acting on central and peripheral neurons.17,18 Independent
studies have shown that GDNF binds to a novel glycosylphosphatidylinositol (GPI)-linked protein (termed GDNFRa) by promoting the formation at the cell surface of a physical complex involving GDNF, GDNFR-a, and RET.19,20 As
EMATOPOIESIS IS A tightly regulated process in
which a small population of self-renewing primitive
progenitors generates an offspring of increasingly differentiated end cells with specific functional activities.1 This process is controlled by a number of growth factors and cytokines,1 with some of them exerting their specific functions
through the binding to high-affinity receptor tyrosine kinases
(RTKs), which are differentially expressed on the various
hematopoietic cell subsets.2,3 RTKs have been shown to act
as important regulators in the processes of growth and differentiation of hematopoietic progenitors. For example, the type
III RTK subfamily includes the product of proto-oncogene
FMS, which was identified as the receptor for macrophage
colony-stimulating factor,4 a critical cytokine for cells of the
monocyte-macrophage1,4 and osteoclast lineages.5 Similarly,
products of KIT and FLT3/FLK2 genes, respectively representing transmembrane receptors for stem cell factor6 and
FLT3-ligand,7,8 are other type III RTKs playing a pivotal
role in the early steps of hematopoiesis and contributing to
the functional regulation of specific cell types, such as
CD34/ progenitors, mast cells, megakaryocytes, and osteoclasts.6,8,9 More recently, a novel RTK, named TNK1, exerting a regulatory role in early hematopoiesis, has been
cloned from human umbilical cord blood CD34/ stem cells.10
In addition, the product of TRK proto-oncogene, encoding
a high-affinity nerve growth factor receptor, although first
described as a nonhematopoietic RTK, has been later shown
to be expressed and functionally active in normal and malignant myelomonocytic cells.11
The RET proto-oncogene encodes for a member of the
RTK superfamily2 whose structure consists of cadherin-like
and cysteine-rich repeats in the extracellular region, a hydrophobic transmembrane domain, and a split intracellular
tyrosine kinase region.12-14 Recently, evidence has been provided that RET may act as a signaling component of a multi-
From the Unità Operativa Leucemie e Trapianto di Midollo, Divisione di Oncologia Medica, Centro di Riferimento Oncologico,
INRCCS, Aviano, Italy; the Dipartimento di Medicina Sperimentale
e Clinica, Facoltà di Medicina e Chirurgia, Università degli Studi
di Reggio Calabria, Catanzaro, Italy; the Centro di Endocrinologia
ed Oncologia Sperimentale del CNR, c/o Dipartimento di Biologia e
Patologia Cellulare e Molecolare, Facoltà di Medicina e Chirurgia,
Università degli Studi di Napoli ‘‘Federico II,’’ Napoli, Italy; and
the Istituto dei Tumori Fondazione ‘‘Pascale,’’ Napoli, Italy.
Submitted May 23, 1996; accepted November 19, 1996.
A. Celetti and A. Cerrato equally contributed to this work.
Supported by the Associazione Italiana per la Ricerca sul Cancro
(Milano, Italy); the Ministero della Università, Ricerca Scientifica
e Tecnologica (Rome, Italy); the Consiglio Nazionale delle Ricerche,
PF-ACRO, Italy; and the Ministero della Sanità, Ricerca Finalizzata
IRCCS (Rome, Italy). Part of this work was performed while G.V.
was holding a position of Fogarty-Scholar-in-Residence at the National Institutes of Health, Bethesda, MD.
Address reprint requests to Michele Grieco, MD, Dipartimento di
Medicina Sperimentale e Clinica, Facoltà di Medicina e Chirurgia,
Università degli Studi di Reggio Calabria, Via T. Campanella, I88100, Catanzaro, Italy.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
q 1997 by The American Society of Hematology.
0006-4971/97/8908-0040$3.00/0
Blood, Vol 89, No 8 (April 15), 1997: pp 2925-2937
AID
Blood 0037
/
5H33$$$721
2925
03-27-97 13:45:36
blda
WBS: Blood
2926
GATTEI ET AL
Fig 1. Validation of competitive RT-PCR strategy by use of a nonhomologous DNA fragment (RETComp ) engineered to contain specific RET
primer templates. (A) Kinetics of amplification of RET cDNA and RETComp fragments. Fixed amounts (0.1 attomoles) of RET cDNA and RETComp
fragments were amplified in a single reaction tube with specific RET primers. After 22 amplification cycles and after each of 8 additional cycles,
a small aliquot of the reaction mixture was removed and the products were resolved on agarose gel (upper panel). The relative intensities of
the bands corresponding to RET cDNA (790 bp) and RETComp (597 bp) -amplified products were quantified by computer imaging. The amount
of specific amplified products for RET cDNA (s) and RETComp (●), expressed in AU, was plotted as a function of the number of cycles (lower
panel). (B) Determination of relative levels of RET mRNA in THP-1 cells by competitive RT-PCR. Ten-fold serial dilutions (10 to 1 Ì 10Ï5
attomoles) of RETComp were amplified with RET primers together with constant aliquots of cDNA from the THP-1 cell line. After 35 cycles,
amplified products were resolved on agarose gel (upper panel). Relative intensities of the bands were densitometrically determined and the
logarithm of their ratios was plotted as a function of the logarithm of the amount of RETComp added (lower panel). The equivalence point
(arrow) was inferred between 10Ï1 and 10Ï2 attomoles.
a result of such interactions, RET-mediated intracellular signaling is activated.19,20
Although previous studies have indicated an important
role for the RET product in the physiologic development and
differentiation of neural crest derivatives, enteric nervous
system, and components of the excretory system,21-25 as well
as its involvment in some forms of human neoplasia,26,27
the role of this RTK in human hematopoiesis has not been
addressed so far in detail. On the other hand, RET transcripts
and/or protein have been detected in hematopoietic fetal
liver22 and in two myeloid leukemic cell lines of human
origin, ie, HL-60 and THP-1,28-30 thus suggesting that some
cells of hematopoietic origin could express the RET-encoded
RTK.
To better understand whether RET receptor might be involved in the regulation of human hematopoiesis, we have
analyzed its levels of expression in purified normal and malignant cells of myeloid and lymphoid lineages mirroring
various stages of hematolymphopoietic differentiation, leukemia/lymphoma cell lines, and stromal cells of the bone
marrow microenvironment. In addition, the presence of
GDNFR-a in the same cell types and the functional effects
of GDNF on human leukemic cells were analyzed.
MATERIALS AND METHODS
Cell samples. The study included cellular samples obtained
from peripheral blood (PB) or bone marrow (BM) of 154 patients
with acute myeloid leukemias (AML; n Å 53); chronic myelopro-
AID
Blood 0037
/
5H33$$$721
liferative disorders in myeloid blast crisis (MBC; n Å 5); B- and
T-cell lymphoproliferations (n Å 96), including B- and T-lineage
acute lymphoblastic leukemias (ALL); chronic lymphocytic leukemia (CLL); prolymphocytic leukemia (PLL); hairy cell leukemia (HCL); high- and low-grade non-Hodgkin’s lymphomas
(NHL) in overt leukemic phase; multiple myeloma (MM); and
adult T-cell leukemia/lymphoma (ATLL). Diagnoses were based
on cell morphology, immunophenotyping, enzyme cytochemistry,
and clinical parameters. Acute leukemias were classified according to the revised French-American-British (FAB) criteria. 31
NHL was diagnosed by histopathologic examination of lymph
node tissues and immunohistochemistry and classified according
to the International Working Formulation.32
Cell isolation and purification. Anticoagulated PB and BM aspirates were collected from leukemia/lymphoma patients after informed consent was obtained and before therapy. Neoplastic cells
were isolated by centrifugation on a Ficoll-Hypaque (Pharmacia,
Uppsala, Sweden) gradient and, with the exclusion of T-cell malignancies, further purified by T-cell depletion with anti-CD2 immunomagnetic beads (Dynabeads; Dynal, Oslo, Norway). In the case of
MM, tumor cells were further purified by positive indirect immunomagnetic selection with the plasma cell-specific monoclonal antibody (MoAb) BB-4.33 After purification procedures were performed,
all of the samples contained more than 95% of neoplastic cells.
Purification of normal cells from PB and tissues was performed
essentially as described.34,35 Briefly, Ficoll-Hypaque–isolated circulating mononuclear cells were further purified by positive immunomagnetic selection with anti-CD2, anti-CD19, anti-CD4, and antiCD8 immunomagnetic beads (Dynal) to obtain, respectively, T and
B lymphocytes as well as CD4/ and CD8/ T-cell subsets. For in
vitro activation studies, purified CD2//CD3/ T lymphocytes were
03-27-97 13:45:36
blda
WBS: Blood
RET AND GDNFR-a IN HUMAN HEMATOPOIESIS
2927
Fig 2. Constitutive expression of RET transcripts (semiquantitative RT-PCR) and GDNFR-a (RT-PCR) in normal lymphohematopoietic
cells. Cell fractions were isolated to purity by discontinuous gradient
centrifugation and/or immunomagnetic selection. Peripheral T cells
were activated with 10 ng/mL TPA plus 1.0 mg/mL of ionomycin A
for 72 hours. Adherent macrophages (Adh. macroph.) were activated
with 100 ng/mL of GM-CSF for 12 hours. In all cases, 1 mg of total
RNA was reverse-transcribed in a 20-mL reaction mix containing
hexadeoxyribonucleotides random primers. Four microliters was amplified with primers specific for RET and GDNFR-a. In the case of
RET, amplification was performed in the presence of a constant
amount (10Ï3 attomoles) of RETComp fragment. After 35 cycles of amplification, 15 mL of PCR products was resolved on 1.5% agarose gel,
blotted, and hybridized with specific RET and GDNFR-a oligoprobes.
For semiquantitative evaluation of RET transcripts, ratios of the relative intensities of bands corresponding to RET cDNA (790 bp) and
RETComp (597 bp) -amplified products were quantified by computer
imaging of gel, expressed in AU, and graphed. An adult human brain
substantia nigra cDNA and cDNA derived from the MN-60 cell line
were respectively used as positive (") and negative (Ï) controls for
RET and GDNFR-a expression. cDNAs were always tested with bactin–specific primers.
exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA; Sigma, St
Louis, MO; 10 ng/mL) plus ionomycin A (Sigma; 1.0 mg/mL) for 72
hours, as reported.34,35 Plastic-adherent macrophages were recovered
after 2 hours of incubation at 377C of the CD20/CD190 PB mononuclear cell (PBMC) fraction. In some experiments, adherent macro-
AID
Blood 0037
/
5H33$$$721
phages were activated in vitro by exposure to 100 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF) for 12 hours.
More than 96% pure neutrophils and eosinophils were isolated from
PB buffy coats by 1.2% dextran sedimentation, followed by a multiple density Percoll gradient centrifugation, as described.36 Tissue T
and B lymphocytes were purified by tearing out single cells from
freshly excised tonsils and thymectomy samples. After Ficoll-Hypaque separation, CD2/, CD4//CD8/, and CD19/ cells were further
purified by immunomagnetic selection. CD34/ hematopoietic progenitors were isolated from PB of patients undergoing high-dose
chemotherapy followed by granulocyte colony-stimulating factor
and from umbilical cord blood by using affinity columns (MACS
CD34 Cell Isolation Kit; Miltenyi Biotec, Celbio, Milan, Italy).36 In
all experiments, purity of the selected cell fractions was estimated
by morphology and flow cytometry by appropriate MoAb combinations.34 BM aspirates, which were obtained after informed consent
from patients with solid tumors undergoing routine staging procedures, were used as a source of BM-derived primary stromal cells
(BMSC) and fibroblasts (BMF). BMSC were obtained from FicollHypaque–separated mononuclear BM cells cultured in Iscove’s
modified Dulbecco’s medium (IMDM) supplemented with 12.5%
fetal calf serum (FCS), 12.5% horse serum (Hyclone, Logan, UT),
and 1.0 1 1006 mol/L hydrocortisone hemisuccinate (Sigma), as
previously described.9 For BMF preparation, hydrocortisone was
substituted for with 0.1 ng/mL basic fibroblast growth factor (bFGF;
Genzyme Co, Cambridge, MA). After four to five passages, cultures
developed in the presence of bFGF were virtually free of contaminating endothelial cells and macrophages, as verified by immunostaining for von Willebrand’s factor, nonspecific esterase, and CD14.9
Cell lines and culture conditions. K562 (early myeloerythroid),
HEL (myeloblastic-erythroblastic), KG-1A (early myeloblasts,
CD34/), KG-1 (early myeloblasts, CD340), HL-60 (intermediate
myeloid-promyelocytes), U937 (early monoblasts), ML3 (myelomonoblasts), THP-1 (monoblasts), Mo7e (megakaryoblast), NB-4
[leukemic promyelocytes harboring the t(15;17) translocation], FLG
29.1 (pre-osteoclasts), Molt-4, FRO (common thymocyte phenotype,
T cells), Jurkat, CEM, KE37 (postthymic phenotype, T cells), H9,
HUT 78, HUT 102, Karpas 299 (mature T-cell phenotype), BV-173
(early B lymphoblasts; lymphoid blast crisis of chronic myelogenous
leukemia), Ci-1, Ri-1, SC-1 (B-cell NHL), Nalm-6 (early pre-B
cells), MN-60 (SIg/ B-cell ALL), HBL-1, HBL-2, HBL-3 (small
noncleaved cell lymphoma from human immunodeficiency virus
[HIV]/ patients), JD38, Namalwa (sporadic and endemic Burkitt
lymphoma), U266, LP1, IM9 (MM), Saos-2, MG-63 (osteoblast
cells), and C433 (derived from the stromal component of a giant
cell tumor of bone) cell lines were cultured in RPMI 1640 medium
(GIBCO, Paisley, UK) supplemented with 10% of FCS, with the
exception of KG-1, HEL, and THP-1, which were maintained in
IMDM (GIBCO) plus 20% FCS and Saos-2 and MG-63, cultured
in McCoy’s medium (GIBCO) supplemented with 10% FCS. Mo7e
cells were cultured in IMDM plus 5% FCS supplemented with 10
ng/mL GM-CSF. Sources and phenotypic characterization of all the
above cell lines have been reported in detail previously.9,34,35,37 U937
cells were induced to differentiate into adherent mature macrophages
by incubation with 1 1 1007 mol/L TPA or vitamin D3 (250 ng/
mL; kindly supplied by Dr J. Hadvary, Hoffmann-La Roche, Basel,
Switzerland) plus transforming growth factor-b (TGF-b; 1.0 ng/mL;
R&D System Europe, Abington, UK).38
RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR). Total RNA (1 mg), extracted by the guanidium
thiocyanate method,39 as well as poly A/ RNA from human adult
brain substantia nigra (Clontech Laboratories Inc, Palo Alto, CA)
were reverse-transcribed by avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI) for 1.5 hours at 427C in a 20-mL
reaction mix containing hexadeoxyribonucleotides random primers
03-27-97 13:45:36
blda
WBS: Blood
2928
GATTEI ET AL
Fig 3. Constitutive expression of RET protein in purified human normal neutrophils, as detected by immunocytochemistry (A and B), and
in CD14" PB monocytes, as detected by Western blotting (C). (A) Neutrophils stained with affinity-purified nonimmune rabbit serum (0.1 mg/
mL). (B) Neutrophils stained with affinity-purified rabbit polyclonal antibodies recognizing the RET tyrosine-kinase domain (0.1 mg/mL). Original magnification for (A) and (B) Ì 400. (C) Western blot analysis of RET
protein in human normal monocytes. Proteins extracted from purified
CD14" PB monocytes, THP-1 (positive control), and undifferentiated
U937 (negative control) cell lines were immunoprecipitated with two
antibodies (Ab 1 and Ab 2) recognizing different cytoplasmic domains
the RET RTK, blotted onto immobilon-P membranes, and shown by
standard chemiluminescence.
(0.4 mg). Four microliters of the same cDNA preparations was amplified in a 50-mL volume of final reaction mix in a Perkin Elmer 9600
thermal cycler (Perkin Elmer Cetus, Emeryville, CA) with 25 pmol
of primers specific for RET (sense, 5*-CAG CTG CTT GTA ACA
GTG-3*, region 1426-1443; antisense, 5*-CTT TCA GCA TCT TCA
CGG-3*, region 2215-2198),12 GDNFR-a (sense, 5*-CGG TTA ACA
GCA GGT TGT CAG A-3*, region 669-690; antisense, 5*-GTG
TGG GGA TCT CAT TCT CAG AC-3*, region 1469-1447),19 and
b-actin (Clontech Laboratories Inc; sense, region 578-609; antisense,
region 1415-1384). Primers pairs were selected spanning different
introns to distinguish amplified cDNA products from genomic DNA.
In the case of RET proto-oncogene, primers amplify a 790-bp region
spanning from the extracellular domain upstream the cysteine-rich
AID
Blood 0037
/
5H33$$$721
domain up to the kinase domain downstream the ATP binding site.12
PCR conditions for RET were 4 minutes at 947C followed by 35
cycles of 45 seconds at 947C, 45 seconds at 627C (587C for GDNFRa), 60 seconds at 727C, and a final extension of 5 minutes at 727C.
In the case of b-actin, amplification was performed for 30 cycles
according to the manufacturer’s guidelines. Fifteen microliters of
amplified cDNAs was run in 1.5% agarose gels, blotted onto nylon
membranes (Boehringer Mannheim, Mannheim, Germany), and hybridized with 2 1 106 cpm/mL of 5* end-labeled oligoprobes specifically designated to recognize PCR products. Probes for RET and
GDNFR-a spanned nucleotide positions 1430-1454 and 708-735,
respectively.12,19
Differences in RET expression in normal and leukemic cells were
03-27-97 13:45:36
blda
WBS: Blood
RET AND GDNFR-a IN HUMAN HEMATOPOIESIS
2929
Table 1. Expression of RET Proto-Oncogene and GDNFR-a in
Human Leukemia/Lymphoma Cell Lines
Expression of
Lineage
Cell Line
Phenotype
RET
GDNFR-a
Myelo-monocytic
KG-1
KG-1A
HL-60
NB-4
U937
ML-3
THP-1
K562
HEL
Mo7e
FLG 29.1
Molt-4
FRO
Jurkat
CEM
KE-37
H9
HUT 78
HUT 102
Karpas-299
BV-173
Nalm-6
MN-60
Ci-1
Ri-1
Sc-1
HBL-1
HBL-2
HBL-3
JD38
Namalwa
U266
LP-1
IM-9
CD340 early myeloblasts
CD34/ early myeloblasts
Myeloblasts-Promyelocytes
Promyelocytes, t(15;17)
Early monoblasts
Myelo-monoblasts
Monoblasts
Early myelo-erythroblasts
Myelo-erythroblasts
Megakaryoblasts
Pre-osteoclasts
Thymic (CD4//CD8/)
Thymic (CD4//CD8/)
Postthymic (CD4//CD80)
Postthymic (CD4//CD80)
Postthymic (CD4//CD80)
Mature T (CD4//CD80)
Mature T (CD4//CD80)
Mature T (CD4//CD80)
Mature T (CD4//CD80)
Early pre-B (SIg0)
Late pre-B (SIg0)
Early B (SIg/)
B-cell lymphoma (SIg/)
B-cell lymphoma (SIg/)
B-cell lymphoma (SIg/)
SNCCL-HIV/ (SIg/)
SNCCL-HIV/ (SIg/)
SNCCL-HIV/ (SIg/)
sBL (SIg/)
eBL (SIg/)
Plasmacell
Plasmacell
Plasmacell
0
/
/
/
0
/
/
0
0
/
0
0
0
/
0
/
/
/
0
/
0
/
0
0
0
0
0
0
0
0
0
0
0
/
0
/
0
0
0
0
0
0
0
0
0
/
0
0
0
0
0
0
0
0
0
/
0
0
0
0
0
0
0
0
0
0
0
0
T cells
B cells
RETComp (10 to 1.0 1 1005 attomoles) were amplified together with
constant aliquots (4 mL) of cDNA from the THP-1 cell line, used
as a positive control for RET transcripts,28-30 and resolved on agarose
gels (Fig 1B, upper panel). Relative intensities of the bands were
densitometrically quantitated by computer imaging, expressed as
arbitrary units (AU) after correction for the size difference between
RET and RETComp , and the logarithms of their ratios were plotted as
a function of the logarithm of the amount of RETComp added (Fig
1B, lower panel). This plot was used to determine the equivalence
point, ie, the point at which the logarithm of the ratio of RET to
RETComp is equal to 0, ie, the amount of RET is equal to the amount
of RETComp . In Fig 1B, the equivalence point was inferred between
1001 and 1002 attomoles (arrows). Owing to the abundance of RETspecific RNA in the THP-1 cell line,28-30 the use of an RETComp
amount about 1 log lower than the equivalence point (ie, 1003 attomoles) was judged to be optimal for further studies of semiquantitative competitive RT-PCR to detect RET transcripts also in samples
with a low expression rate. For such studies, constant amounts of
RETComp were amplified by RET-specific primers in the same tube
together with 4 mL of reverse-transcribed cDNA from the experimental cell samples in a final 50- mL volume of reaction mix. After
resolution on agarose gels, band intensities were quantitated by computer imaging and the relative AU ratios were calculated. Differences
in these ratios indicated the relative differences in mRNA levels
among the different samples.
Western blot and immunoprecipitation. Anti-RET antibodies included a rabbit polyclonal antibody raised against the entire RET
tyrosine kinase domain (Ab 1) and a rabbit polyclonal antibody
raised against a carboxyterminal peptide (residues 1011-1027) of the
cytoplasmic domain of human RET (Ab 2),42 both purified by affinity
chromatography. Immunoprecipitation and immunoblotting experiments using anti-RET antibodies were performed as previously
RET expression was detected by RT-PCR.
Abbreviations: GDNFR-a, glial cell line-derived neurotrophic factor; SIg, surface
Ig; SNCCL, small noncleaved cell lymphoma; sBL, sporadic Burkitt’s lymphoma;
eBL, endemic Burkitt’s lymphoma.
evaluated by a quantitative and semiquantitative RT-PCR approach.
For this purpose, an internal competitor (RETComp ), with a different
size than RET-specific amplicons, was prepared from an unrelated
DNA fragment engineered to contain specific RET primer templates
essentially as described.40,41 Briefly, a BamHI/EcoRI v-erb B 580bp DNA fragment (Clontech Laboratories Inc) was amplified first
with composite primers, containing both RET- and v-erb B-specific
sequences, and then with RET gene-specific primers alone. This
procedure gave rise to a 597-bp nonhomologous DNA fragment
(RETComp ) containing at its ends the appropriate templates for RET
primers. RETComp fragments, when amplified with RET-specific primers, yielded to a 597-bp band that was easily discriminated from the
790-bp RET-related amplicon on 1.5% agarose ethidium bromidestained gels. After purification and densitometric quantitation, a
comparison between the amplification kinetics of purified RET amplicons (obtained from THP-1 cells cDNA) and RETComp was performed by amplifying in the same tube 0.1 attomoles of each fragment, removing 5-mL aliquots of reaction mix after 22 to 30 cycles,
and analyzing them separately on agarose gels (Fig 1A). A stepwise
increase of specific amplified products corresponding to RET (790
bp) and RETComp (597 bp) was observed between 22 and 30 amplification rounds (Fig 1A, upper panel). Quantitation of RET- and
RETComp -specific bands by gel analyzer (Gel Doc 1000, BioRad
Laboratories, Hercules, CA) resulted in two exponential curves with
comparable slopes (Fig 1A, lower panel), indicating a similar amplification efficiency for both fragments. To determine the optimal
amount of RETComp for screening studies, 10-fold serial dilutions of
AID
Blood 0037
/
5H33$$$721
Fig 4. Constitutive expression of RET transcripts (semiquantitative RT-PCR) and GDNFR-a (RT-PCR) in human leukemia/lymphoma
cell lines. One microgram of total RNA was reverse-transcribed in
a 20-mL reaction mix containing hexadeoxyribonucleotides random
primers. Conditions for RT-PCR, blotting, hybridization, semiquantitative analysis, and controls were as described in Fig 2.
03-27-97 13:45:36
blda
WBS: Blood
2930
GATTEI ET AL
Table 2. Expression of RET Proto-Oncogene and GDNFR-a in AML
Expression of
Diagnosis
RET
(no. positive/no. tested)
AML
M0
M1
M2
M3
M4
M5
M6
M7
MBC
CML
PV
IM
GNDFR-a
(no. positive/no. tested)
32/53
0/3
5/13
3/4
6/7
10/14
7/10
0/1
1/1
1/42
0/2
0/10
0/3
0/5
0/12
0/8
0/1
1/1
3/5
2/3
0/1
1/1
0/4
0/2
0/1
0/1
RET expression was detected by RT-PCR.
Abbreviations: GDNFR-a, glial cell line-derived neurotrophic factor; MBC, myeloid blast crisis of myeloproliferative disorders; CML, chronic myeloid leukemia;
PV, polycythemia vera; IM, idiopathic myelofibrosis.
described.43 Briefly, cells were lysed in 50 mmol/L N-2-hydroxyethylpiperazine-N*-2-ethanesulfonic acid, pH 7.5, 1% (vol/vol) Triton
X-100, 50 mmol/L NaCl, 5 mmol/L EGTA, 50 mmol/L NaF, 20
mmol/L sodium pyrophosphate, 1 mmol/L sodium vanadate, 2
mmol/L phenylsulphonyl fluoride, and 0.2 mg each of aprotinin and
leupeptin per milliliter. Lysates were clarified by centrifugation at
10,000g for 15 minutes and the supernatants were processed for
immunoprecipitation as described.43 Immunoblots, after incubation
with the appropriate antibodies, were shown with the Amersham
ECL (Amersham Co, Amersham, UK) system.
Treatment of cells with phosphatidylinositol-specific phospholipase C (PI-PLC) and preparation of a BMSC-derived PI-PLC conditioned medium (PI-PLC/CM). To release GPI-anchored proteins
from the cell membrane, primary BMSC, BMF, and the MG-63
osteoblast cell line were preincubated for 1 hour at 377C with 1 U/
mL of PI-PLC19,20 (Boehringer Mannheim), washed three times in
IMDM, and then used for flow cytometry analysis (see below). To
obtain PI-PLC/CM, 5 to 10 1 106 of primary BMSC were incubated
in 1 mL of culture medium in the presence of 1 U/mL of PI-PLC
as described above.19 After the removal of cells by centrifugation,
the PI-PLC/CM was collected and immediately used for colony assay
and flow cytometry.
Leukemic blast colony assay and liquid cultures. The number of
leukemic colony-forming units (CFU-L) was assessed as previously
described.44 Briefly, 1.0 1 105 T-cell–depleted leukemic blasts were
resuspended in 1 mL of IMDM containing 20% FCS and 0.8%
methylcellulose and cultured in 100-mL aliquots (6 to 8 replicates)
in 96-well flat-bottomed microplates in the presence of increasing
concentrations (0.5 to 10 ng/mL) of GDNF (Promega). When indicated, freshly prepared BMSC-derived PI-PLC/CM was added to
semisolid medium (10% vol/vol) immediately before plating. After
7 to 14 days of incubation, aggregates with ¢40 cells were scored
as colonies.
For differentiation studies, leukemic blast cells (2 1 105/mL) were
incubated in the presence of a mixture of GDNF (10 ng/mL) and
stromal cell-derived PI-PLC/CM (10% vol/vol), recovered from liquid cultures at different time points, and morphologically analyzed
by May-Grünwald-Giemsa staining of cytospin preparation. The percentage of adherent cells was evaluated by scoring the number of
cells that needed trypsinization to be detached from the plates compared with the number of cells growing in suspension, as previously
described.45 Control experiments were performed by incubating cells
either in the presence of GDNF without PI-PLC/CM or with PIPLC/CM alone.
Immunocytochemistry and flow cytometry. Immunocytochemistry procedures were performed, following the manufacturer’s guidelines, with a modified avidin-biotin-complex technique (Kirkegaard & Perry Laboratories Inc, Gaithersburg, MD) on cytospin
preparations of purified normal granulocytes, CD14/ monocytes,
and selected AML cases. Cytospin slides were fixed in 4% paraformaldehyde plus 0.5% Triton in Tris-HCl buffer and sequentially
incubated with affinity-purified rabbit polyclonal antibodies (0.1 mg/
mL) recognizing the RET tyrosine-kinase domain,42,43 biotinylated
goat antirabbit IgG, and finally alkaline phosphatase-conjugated
streptavidin. Immunostaining was developed by incubating the slides
with the appropriate chromogenic substrates (Kirkegaard & Perry
Laboratories Inc) followed by hematoxylin (Sigma) counterstain.
Controls included the omission of the primary antibody and the use
of affinity-purified nonimmune rabbit serum (0.1 mg/mL). For flow
Fig 5. Constitutive expression of RET transcripts and GDNFR-a in leukemic cells of myeloid origin (left panels) and in malignant cells from
lymphoid tumors (right panels), as assessed by RT-PCR. One microgram of total RNA from AML cases (identified according to their FAB
classification) and various lymphoid tumors was reverse-transcribed and amplified with primers specific for RET and GDNFR-a. PCR products
were resolved on agarose gel, blotted, and hybridized with specific RET and GDNFR-a oligoprobes. An adult human brain substantia nigra
cDNA and cDNA derived from the MN-60 cell line were used, respectively, as positive (") and negative (Ï) controls for RET and GDNFR-a
expression. cDNAs were always tested with b-actin–specific primers.
AID
Blood 0037
/
5H33$$$721
03-27-97 13:45:36
blda
WBS: Blood
RET AND GDNFR-a IN HUMAN HEMATOPOIESIS
2931
cytometric detection of surface GPI-linked GDNF receptors
(GDNFR-a),20 BMSC, BMF, and MG-63 cells, pretreated or not
with PI-PLC as described above, were exposed for 1 hour at 377C
with GDNF (100 ng/mL; Promega) and then incubated sequentially
with chicken polyclonal antihuman GDNF (100 mg/mL; Promega)
and rabbit fluorescein isothiocyanate-conjugated isotype-matched
antichicken Igs (Promega). As controls, isotype-matched irrelevant
chicken Igs (Promega) were used. In some experiments, primary
leukemic cells were exposed for 1 hour at 377C to GDNF alone (100
mg/mL) or to a combination of GDNF and BMSC-derived PI-PLC/
CM, before being processed for flow cytometric detection of membrane-bound GDNF. Viable, antibody-labeled cells were identified
according to their forward and side scatter, electronically gated,
and assayed for surface fluorescence on a FACScan flow cytometer
(Becton Dickinson, Mountain View, CA).
RESULTS
Expression of RET proto-oncogene and GDNFR-a by normal hematolymphoid cells. cDNAs obtained from purified
cell populations were assayed for RET expression by semiquantitative RT-PCR and the specificity of amplified products was confirmed by Southern blotting hybridization with
an internal RET oligoprobe. As shown in Fig 2, a faint 790bp band corresponding to RET amplified products was detected in CD34/ hematopoietic progenitors from mobilized
PB and cord blood (Fig 2). As compared with early progenitors, higher levels of RET transcripts were found in circulating neutrophils (5-fold) and adherent CD14/ monocyte/macrophages (6-fold), being further increased (11-fold) in these
latter cells upon GM-CSF–induced cellular activation (Fig
2). In contrast, circulating eosinophils and different T-cell
subsets from PB, thymus, and tonsils did not express RETspecific mRNA, which remained undetectable in normal peripheral T cells also after cellular activation by TPA and
ionomycin (Fig 2). Similarly, tonsil and PB CD19/ B cells
were negative for RET mRNA (Fig 2). As opposed to RET
proto-oncogene, transcripts specific for GDNFR-a were
never found in all the normal cell types analyzed (Fig 2).
In agreement with RT-PCR data, expression of RET protein was detected by immunocytochemistry and Western
blotting both in purified neutrophils and in CD14/ peripheral
monocytes (Fig 3 and data not shown). In the case of neutrophils, a prominent granular staining of cytoplasms, along
with a faint membrane reactivity in some of the cells, was
usually observed (Fig 3B), whereas in monocytes a strong
and diffuse cytoplasmic staining was commonly associated
with a clear membrane labeling (data not shown). As shown
in Fig 3C, RET products of 150 and 170 kD were detected
in CD14/ purified monocytes by Western blotting with two
different anti-RET polyclonal antibodies (Ab 1 and Ab 2).
In agreement with previous data,30 RET-specific components
of 150 and 190 kD were found with both antibodies in the
THP-1 cell line, which was used as a positive control,
whereas no RET protein was immunodetected in undifferentiated U937 cells (Fig 3C).
Expression of RET proto-oncogene and GDNFR-a by human leukemic cell lines of myeloid and lymphoid lineages.
By studying a large panel of human leukemia/lymphoma cell
lines, RET mRNA was detected, albeit at different constitutive levels, in malignant cells of both myeloid and lymphoid
AID
Blood 0037
/
5H33$$$721
Table 3. Expression of RET Proto-Oncogene in Human Lymphoid
Hematopoietic Malignancies
Expression of
Diagnosis
B-cell tumors
B-lineage ALL
B-CLL
B-PLL
HCL
B-NHL
HG
LG
MM
T-cell tumors
T-ALL
T-PLL
ATLL
RET
(no. positive/no. tested)
GNDFR-a
(no. positive/no. tested)
13/85
2/7
5/45
0/2
1/7
0/60
0/6
0/30
0/1
0/5
2/9
2/9
1/6
3/11
2/9
0/1
1/1
0/6
0/6
0/5
1/8
0/6
0/1
1/1
RET expression was detected by RT-PCR.
Abbreviations: GDNFR-a, glial cell line-derived neurotrophic factor;
CLL, chronic lymphoblastic leukemia; PLL, prolymphocytic leukemia;
HCL, hairy cell leukemia; HG, high grade; LG, low grade; ATLL, adult
T-cell leukemia/lymphoma.
origin. As shown in Table 1 and Fig 4, the highest relative
levels of RET transcripts were detected in cell lines of the
myelomonocytic lineage, whereas most tumor B-cell lines,
encompassing early pre-B to plasmacell differentiation
stages, were either negative (11/13) or displayed (Nalm-6
and IM-9) low levels of RET mRNA. Among cell lines of
myelo-granulocytic (KG-1, KG-1A, HL-60, and NB-4) and
monocytic (U937, ML-3, and THP-1) phenotypes, a correlation between the constitutive expression of RET proto-oncogene and the relative maturation stage was observed (Fig 4).
In particular, the early (CD34/) myeloblasts cell line KG1A displayed low levels of RET mRNA, which were more
than threefolds and greater than fivefold higher in the intermediate myeloblast cell line HL-60 and in the promyelocytic
leukemia cell line NB-4, respectively (Fig 4). Similarly, the
highest amount of RET transcripts among monocytic cell
lines was detected in those (THP-1) displaying the more
mature phenotype,34 as compared with ML-3 and undifferentiated U937 (Fig 4). Accordingly, a significant upregulation
of RET mRNA levels was observed by semiquantitative RTPCR upon induction of monocytic maturation of U937 cells
by either TPA (8-fold at day 3) and vitamin D3 plus TGFb (23-fold at day 6), along with a parallel increase of immunodetectable RET protein (data not shown). No RET expression was detected in cell lines of erythroid derivation (K562
and HEL) and osteoclast phenotype (FLG 29.1), whereas
very low levels of RET transcripts were found in the megakaryocytic cell line Mo7e (Fig 4). Although the relative
amount of RET transcripts detected in human leukemia/
lymphoma T-cell lines was overall lower than in myeloid
cell lines, a correlation between the levels of RET expression
and the relative maturation stage again emerged from our
analysis (Fig 4). As shown by semiquantitative RT-PCR
assay, cell lines of mature T-cell phenotype (H9, HUT 78,
and Karpas-299) displayed RET mRNA levels about fivefold
03-27-97 13:45:36
blda
WBS: Blood
2932
GATTEI ET AL
Fig 6. (A) RT-PCR detection of RET and GDNFR-a mRNA in primary BMSC from two donors (nos. 1 and 2), BMF, Saos-2, and MG-63
osteoblast cell lines and C433 stromal cell line. Aliquots of cDNA bulks were amplified with primer pairs specific for RET and GDNFR-a, run
in agarose gels, blotted, and hybridized with oligoprobes specifically designated to recognize PCR-amplified products. An adult human brain
substantia nigra cDNA and cDNA derived from the MN-60 cell line were used, respectively, as positive (") and negative (Ï) controls for RET
and GDNFR-a expression. cDNAs were always tested with b-actin–specific primers. (B) Fluorescence histograms showing expression of surface
GPI-linked GDNF receptors (GDNFR-a) in BMSC, BMF, and the MG-63 cell line. Cells pretreated or not with PI-PLC (1 U/mL for 1 hour at 377C)
were incubated for 1 additional hour with GDNF (100 ng/mL) and then sequentially with chicken polyclonal antihuman GDNF (100 mg/mL)
and rabbit fluorescein isothiocyanate-conjugated isotype-matched antichicken Igs. As controls, isotype-matched irrelevant chicken Igs were
used.
higher than cell lines of postthymic phenotype (Jurkat and
KE-37), whereas no expression of the proto-oncogene was
found in cell lines (Molt-4 and FRO) of thymic phenotype
(Table 1 and Fig 4). As compared with RET, the expression
of GDNFR-a was very infrequent, being confined to isolated
cell lines of myelomonocytic cell (KG-1A), T-cell (Molt-4),
and B-cell (Nalm-6) phenotypes (Table 1 and Fig 4).
Expression of RET proto-oncogene and GDNFR-a by primary leukemic cells of myeloid or lymphoid lineages. A
broad expression of the RET proto-oncogene was found in
AML, with about 60% of cases (32/53) displaying significant
amounts of RET RNA (Table 2 and Fig 5). In particular, a
very high frequence of RET expression (17/24 [71%]) was
detected among the monocytic and myelomonocytic subtypes of AML (FAB M4 and M5) as compared with immature myeloid phenotypes (FAB M0 and M1), in which RETpositive samples accounted for 31% (5/16) of cases (Table
2 and Fig 5). In addition, almost all cases (6/7) of acute
promyelocytic leukemia (FAB M3) and FAB M2 AMLs (3/
4) displayed RET transcripts, which were also detected in 3
of 5 samples of MBC (Table 2). Accordingly, expression of
RET protein was detected by immunocytochemistry of blast
cells from selected RET-expressing AML cases (2 FAB-M3,
5 FAB-M4, and 3 FAB-M5), showing a strong cytoplasmic
staining usually associated with a clear membrane labeling
in monocytic-oriented cytotypes (data not shown). Among
AID
Blood 0037
/
5H33$$$721
neoplasms of lymphoid origin (Table 3 and Fig 5), RET
expression was a very infrequent event, being detected in 2
of 9 cases of T-ALLs, in a single case of ATLL (CD4//
CD80), in 2 of 7 samples of B-precursor ALL, and in scattered cases of B-CLL (5/45), HCL (1/7), high-grade (2/9)
or low-grade (2/9) NHL in overt leukemic phase, and MM
(1/6).
As seen in normal hematopoietic cells and leukemic cell
lines, the broad expression of RET proto-oncogene in primary tumor cells was not paralleled by a mutual expression
of GDNFR-a mRNA. In our series, only two isolated cases
(1 AML and 1 T-PLL) were found to express GDNFR-a –
specific transcripts (Tables 2 and 3 and Fig 5).
Expression of RET proto-oncogene and GDNFR-a by BM
stromal cells. The broad expression of RET tyrosine kinase
by normal and malignant hematopoietic cells in the absence
of the naturally occurring receptor for GDNF prompted us
to look for other possible sources of GDNFR-a within the
accessory cells of the hematopoietic microenvironment. As
shown in Fig 6A and B, a high amount of GDNFR-a transcripts and GDNF-binding sites was found in normal BMSC
from two different donors (nos. 1 and 2 in Fig 6A), BMF,
and, albeit at a lower level, two osteoblast cell lines (Saos2 and MG-63) previously described to support normal hematopoiesis.46 The binding of GDNF to stromal cells was virtually abolished after treatment of cells with PI-PLC (Fig 6B),
03-27-97 13:45:36
blda
WBS: Blood
RET AND GDNFR-a IN HUMAN HEMATOPOIESIS
2933
Fig 7. (A) Effects of GDNF alone (right panel) or in association
with a fixed concentration (10% vol/vol) of conditioned medium derived from stromal cells exposed to PI-PLC (PI-PLC/CM; left panel) on
clonogenic growth of blast cells derived from 6 RET-expressing AML
samples. T-cell–depleted leukemic blasts (1.0 Ì 105) were resuspended in 1 mL of IMDM containing 20% FCS and 0.8% methylcellulose and cultured in 100-mL aliquots in 96-well flat-bottomed microplates in the presence of increasing concentrations (0.5 to 10 ng/
mL) of GDNF. Freshly prepared BMSC-derived PI-PLC/CM was added
to semisolid medium (10% vol/vol) immediately before plating. After
7 days of incubation, aggregates with ı40 cells were scored as colonies. Results are expressed as mean Ô SEM of 6 to 8 replicates.
(B) Fluorescence histograms showing the expression of membraneassociated GDNF/soluble GDNF receptors complexes by primary
RET-expressing AML cells. Cells were exposed for 1 hour at 377C to
GDNF alone (100 mg/mL) or to a combination of GDNF and BMSCderived PI-PLC/CM and then sequentially with chicken polyclonal
antihuman GDNF (100 mg/mL) and rabbit fluorescein isothiocyanateconjugated isotype-matched antichicken Igs. As controls, isotypematched irrelevant chicken Igs were used.
which specifically removes GPI-linked molecules from the
cell surface. Interestingly, the expression of GDNFR-a
mRNA in primary stromal cells and stromal cell lines was
not associated with the presence of RET, which was conversely strongly expressed in the absence of GDNFR-a transcripts by another cell line (C433)9 derived from the stromal
component of an osteoclastoma (Fig 6A).
Effects of GDNF on clonogenic growth and monocytic
differentiation of human leukemic cells. Because RET-expressing AML cells do not produce the ligand binding component for GDNF, we have performed functional experiments by using conditioned media from stromal cells as a
putative source of soluble GDNFR-a and/or other GPIlinked GDNF receptors. To this end, T-lymphocyte–depleted mononuclear cells from 6 RET-positive AMLs of myelomonocytic phenotype (FAB M4 and M5) were exposed
at various concentrations of GDNF (0.5 to 10 ng/mL) in a
AID
Blood 0037
/
5H33$$$721
standard clonogenic assay in the presence or not of a fixed
concentration (10% vol/vol) of BMSC-derived PI-PLC/CM.
As shown in Fig 7A (left panel), GDNF, when associated
to BMSC-derived PI-PLC/CM, induced a dose-dependent
impairment of leukemic blasts clonogenic growth, yielding
more than 80% to 90% inhibition of CFU-L at a concentration of 10 ng/mL (Fig 6A). Conversely, in the absence of
BMSC-derived PI-PLC/CM, GDNF-mediated effects of leukemic cell growth were strikingly reduced (Fig 6A, right
panel). The inhibitory effects exerted by GDNF alone on
RET-expressing leukemic cells were overall comparable to
those observed in 3 cases of RET-negative AML cells grown
either in the presence or not of BMSC-derived PI-PLC/CM
(data not shown). The ability of GDNF to bind RET-expressing primary leukemic cells was assessed by a flow cytometric
staining with polyclonal anti-GDNF antibodies in the presence or not of BMSC-derived PI-PLC/CM. Our results indicated that, in agreement with clonogenic data, GDNF was
able to efficiently bind leukemic cells in the precence of
BMSC-derived PI-PLC/CM (Fig 7B). GDNF binding to primary leukemic cells was not increased in the presence of
PI-PLC/CM derived from cells not expressing GDNFR-a
mRNA and used as internal negative control for these experiments (data not shown), suggesting that GDNFR-a could
actually represent the major GDNF-binding activity produced by stromal cells.
To better clarify mechanisms underlying GDNF growth
inhibitory effects on primary AML cells, T-cell–depleted
RET-expressing leukemic blasts (2 AML FAB M4 and 1
AML FAB M5) were exposed in liquid culture to 10 ng/mL
of GDNF in the presence or absence of BMSC-derived PIPLC/CM. As shown in Fig 8, the concurrent exposure of
leukemic blasts to GDNF and BMSC-derived PI-PLC/CM
resulted in a time-dependent increase in the number of cells
adhering to the plastic (Fig 8A and B) and displaying, after
5 days of culture, morphologic changes consistent with differentiation towards mature monocyte/macrophages (Fig 8C
and D). Exposure of blast cells to GDNF alone or BMSCderived PI-PLC/CM alone did not induce significant changes
in the adherence (Fig 8A) and the morphologic appearance
of leukemic cells (not shown). An increased expression of
CD14, CD11b, and CD15 antigens was also found in leukemic cells after exposure to GDNF and BMSC-derived PIPLC/CM (not shown).
DISCUSSION
A putative role of the RET-encoded RTK in the functional
regulation of hematopoietic cells was suggested by the presence of RET transcripts and protein in lympho-hematopoietic
tissues of mice and rats, including fetal liver, thymus, spleen,
and lymph nodes,22,47,48 and in two human leukemic cell lines
(HL-60 and THP-1).28-30 Despite these preliminary indications, expression of the RET RTK in human lymphohematopoietic cells has not been investigated in detail so far.
In the present study, we have shown that relatively low
amounts of RET mRNA can be found in early CD34/ hematopoietic progenitors, but RET transcripts appeared to increase with maturation along the myelomonocytic lineage,
being upregulated in circulating neutrophils and resting or
03-27-97 13:45:36
blda
WBS: Blood
2934
GATTEI ET AL
Fig 8. (A) Adherence of leukemic cells from 3 RETexpressing AML samples cultured in medium alone
(open symbols) or in the presence of a mixture of
GDNF (10 ng/mL) and BMSC-derived PI-PLC/CM
(10% vol/vol) (solid symbols). As further controls,
the percentage of adherent cells was also evaluated
in cultures performed for 5 days in the presence of
GDNF alone (solid symbols at the far right) and
BMSC-derived PI-PLC/CM alone (open symbols at
the far right). The percentage of cells adhering to
plastic dishes was was evaluated by scoring the
number of cells that needed trypsinization to be detached from plates, compared with the number of
cells growing in suspension. Results are mean Ô
SEM of triplicate cultures. (B) Phase-contrast micrograph of differentiated blasts from a 5-day-old culture performed in the presence of GDNF (10 ng/mL)
and BMSC-derived PI-PLC/CM (10% vol/vol) (original
magnification Ì 200). (C and D) Morphologic appearance of leukemic blasts from a RET-expressing AML
(FAB-M5) sample cultured for 5 days in the absence
(C) or in the presence of GDNF (10 ng/mL) and BMSCderived PI-PLC/CM (10% vol/vol) (D); May-GrünwaldGiemsa staining of cytospin preparation (original
magnification for [C] and [D] Ì 630).
AID
Blood 0037
/
5h33$$0037
03-27-97 13:45:36
blda
WBS: Blood
RET AND GDNFR-a IN HUMAN HEMATOPOIESIS
2935
activated monocytes. These results were confirmed at a protein level by immunostaining and Western blotting. Analysis
of human myelomonocytic cell lines was consistent overall
with the pattern of normal cells, showing a progressive increase of RET transcripts in cells representing early to late
stages of granulocytic and monocytic differentiation,34 being
further upregulated during in vitro maturation of monoblastic
cell lines. Accordingly, we have shown that RET expression
is mainly confined to AML displaying either a monocytic
(FAB M4 and M5) or an intermediate-mature myeloid (M2
and M3) phenotype, being less frequently detected in early
myeloid (M0 and M1) AMLs. Taken together, our results
indicate that RET expression is maturation-associated in human myelopoiesis, suggesting a possible role of RET product
in the functional regulation of intermediate and mature myelomonocytic cells. In this regard, RET behavior appeared
to diverge from that of most hematopoietic TRKs, including
KIT, FLT3, TIE, and TNK1, which are usually expressed at
a very high level in early CD34/ progenitors and downregulated or switched off after maturation towards granulocytes
and/or monocytes.2,10,49-51
In contrast, by analyzing normal and malignant B cells
encompassing different stages of B-cell maturation, RET
transcripts were only sporadically detected in SIg/ B-cell
tumors (5/72). These results are in overall agreement with
data from Wasserman et al52 showing that, in mouse B cells,
RET is expressed only in early stages of B-cell differentiation, being drastically downregulated after the expression of
surface Igs. However, we were not able to detect RET mRNA
in most early B-lineage ALLs of pro-B and pre-B phenotypes. The discrepant behavior of RET in the early steps of
mouse and human B-cell lymphopoiesis closely parallels
that of the KIT-encoded RTK, which, although functionally
expressed in mouse early B cells (from pro-B to late preB stage),53,54 is not usually detectable in human B-lineage
ALLs.55
As seen for B cells, purified normal T cells from PB,
tonsil, and thymus never expressed RET proto-oncogene,
even after cellular activation. However, a significant amount
of RET mRNA was detected in some tumor cells of postthymic and mature T-cell phenotype, most of them derived from
dermatotropic T-cell malignancies, including cutaneous Tcell lymphomas (H9, HUT 78),35 CD30/ anaplastic large-cell
lymphoma (Karpas-299),56 and ATLL.57 One can therefore
speculate that the expression of RET RTK may be somehow
associated with cell types characterized by a high migratory
ability, such as neural crest elements,21,22,28,47,48,58 and, as
shown here, monocytes, neutrophils, and dermatotropic malignant T cells.
The expression of the RET gene in normal and malignant
hematopoietic cells raises the question of the functional significance of this RTK in the human hematopoietic compartment,59 in addition to its previously defined role in the regulation of developing central and peripheral nervous system and
kidney.21-25 It has been recently shown that RET RTK is
involved in the formation at the cell surface of a complex
receptor system for GDNF,15,16 in which a GPI-linked molecule (GDNFR-a) acts as a ligand-binding component and
RET represents the signaling component.19,20 It appears there-
AID
Blood 0037
/
5H33$$$721
fore that RET-expressing cells may transduce GDNF-mediated signals in the presence of GDNFR-a.19,20 However, by
analyzing a large variety of purified normal cells, leukemic
cell lines, and primary leukemia/lymphoma cells of myeloid
and lymphoid origin, we were unable to show a consistent
expression of GDNFR-a in human hematopoietic cells, irrespective of the presence of the RET RTK. Because GDNFRa is an extracellular protein that is attached to the outer cell
membrane19,20 and RET activation can be induced by GDNF
in cells not expressing GDNFR-a in the presence of culture
media containing soluble GDNFR-a,19,20 we speculated that
accessory cells of the BM microenvironment could provide
a physiologic source of GDNFR-a for RET-expressing hematopoietic cells. In agreement with such a view, we were
able to show that human BMSC, BMF, and other stromal
cell lines capable of supporting normal hematopoiesis6,46 do
not express RET but produce high levels of GDNFR-a
mRNA and surface GPI-linked GDNF receptors, most probably including GDNFR-a. Accordingly, we have provided
evidence that RET-expressing human AML cells are able to
efficiently bind exogenous GDNF in the presence of supernatants derived from stromal cells treated with PI-PLC to remove GPI-linked proteins (PI-PLC/CM) and that GDNF reduces the clonogenic capacity of leukemic cells in the
presence of stromal cell-derived PI-PLC/CM. In addition,
we have shown that PI-PLC/CM from cells not producing
GDNFR-a did not increase binding and biologic effects of
GDNF on RET-expressing leukemic cells and that clonogenic ability of RET-negative AML cells is not significantly
modified by GDNF, also in the presence of stromal cellderived PI-PLC/CM. These data overall support the idea that
GDNFR-a or a similar, yet unidentified, GPI-linked receptor
for GDNF produced by cells of the BM microenvironment
can mediate the action of this neutrotrophin on human leukemic cells. The mechanisms underlying the inhibitory effects
of GDNF on human AML cells are still obscure, but our
present results seem to indicate that GDNF may induce terminal division and differentiation of monocytic leukemia
cells. Accordingly, we have obtained data that GDNF is able
to enhance both the generation and maturation of monocyte/
macrophage precursors (CFU-M) from normal BM CD34/
cells in the presence of stromal cell-derived PI-PLC/CM
(Gattei et al, manuscript in preparation). Such a possibility
is in agreement with the previous demonstration that GDNF
is able to induce morphologic and functional differentiation
of neural and developing renal cells.17,23-25 The involvement
of GDNF in the functional regulation of normal and neoplastic monocytic cells is also supported by the close developmental relationships between glial cells and the monocyte/
macrophage system60 and by the high levels of expression
of this neurotrophic factor in nonneural tissues, including
hematopoietic organs such as liver and spleen.19,20,61
In light of the emerging role of neurotrophins in the regulation of hematopoietic and immune cells62,63 and of the documented involvement of the RET RTK in a number of neoplastic and nonneoplastic human diseases,26,27,42,58 we are
currently investigating the relationships between accessory
cell-derived GDNFR-a –, GDNF-, and RET-expressing cells
within the normal and neoplastic lymphohematopoietic microenvironment.
03-27-97 13:45:36
blda
WBS: Blood
2936
GATTEI ET AL
ACKNOWLEDGMENT
We are grateful to Dr N. Dathan for providing RET antibodies
and to Dr L. De Marco (Blood Transfusion Center, C.R.O. Aviano,
Aviano, Italy) for kindly providing PB apheresis products. We also
gratefully acknowledge the excellent assistance of Fulvio Coletto
for the artwork and graphic support.
REFERENCES
1. Morrison SJ, Uchida N, Weissman IL: The biology of hematopoietic stem cells. Annu Rev Cell Dev Biol 11:35, 1995
2. van der Geer P, Hunter T, Lindberg RA: Receptor proteintyrosine kinases and their signal transduction pathways. Annu Rev
Cell Biol 10:251, 1994
3. Fantl WJ, Johnson DE, Williams TW: Signalling by receptor
tyrosine kinases. Annu Rev Biochem 62:453, 1993
4. Scherr CJ, Rettenmier CW, Sacca R, Roussel MF, Look AT,
Stanley ER: The c-fms proto-oncogene product is related to the
receptor for the mononuclear phagocyte growth factor, CSF-1. Cell
41:665, 1985
5. MacDonald BR, Mundy GR, Clark S, Wang EA, Kuehl TJ,
Stanley ER, Roodman GD: Effects of human recombinant CSF-GM
and highly purified CSF-1 on the formation of multinucleated cells
with osteoclast characteristics in long-term bone marrow cultures. J
Bone Miner Res 1:227, 1986
6. Galli SJ, Zsebo KM, Geissler EN: The kit ligand, stem cell
factor. Adv Immunol 55:1, 1994
7. Small D, Levenstein M, Kim E, Carow C, Amin S, Rockwell
P, Witte L, Burrow C, Ratajczak MZ, Gewirtz AM, Civin CI: STK1, the human homolog of Flk-2/Flt-3, is selectively expressed in
CD34/ human bone marrow cells and is involved in the proliferation
of early progenitor/stem cells. Proc Natl Acad Sci USA 91:459,
1994
8. Lyman SD, James L, Johnson L, Brasel K, de Vries P, Escobar
S, Downey H, Splett RR, Beckmann MP, McKenna HJ: Cloning of
the human homologue of the murine flt3 ligand: A growth factor
for early hematopoietic progenitor cells. Blood 83:2795, 1994
9. Gattei V, Aldinucci D, Quinn JMW, Degan M, Cozzi M, Perin
V, De Iuliis A, Juzbasic S, Improta S, Athanasou NA, Ashman
LK, Pinto A: Human osteoclasts and preosteoclast cells (FLG 29.1)
express functional c-kit receptors and interact with osteoblasts and
stromal cells via membrane-bound stem cell factor. Cell Growth
Diff 7:753, 1996
10. Hoehn GT, Stokland T, Amin S, RamıD rez M, Hawkins AL,
Griffin CA, Small D, Civin CI: Tnk1: A novel intracellular tyrosine
kinase gene isolated from human umbilical cord blood CD34//Lin0/
CD380 stem/progenitor cells. Oncogene 12:903, 1996
11. Ehrhard PB, Ganter U, Bauer J, Otten U: Expression of functional TRK proto-oncogene in human monocytes. Proc Natl Acad
Sci USA 90:5423, 1993
12. Takahashi M, Buma Y, Iwamoto T, Inaguma Y, Ikeda H,
Hiai H: Cloning and expression of the RET proto-oncogene encoding
a tyrosine kinase with two potential transmembrane domains. Oncogene 3:571, 1988
13. Schneider R: The human protooncogene RET: A communicative cadherin? Trends Biochem Sci 17:468, 1992
14. Kuma K, Iwabe N, Miyata T: Motifs of cadherin- and fibronectin type III-related sequences and evolution of the receptor-typeprotein tyrosine kinases: Sequence similarity between proto-oncogene RET and cadherin family. Mol Biol Evol 10:539, 1993
15. Trupp M, Arenas E, Fainzilber M, Nilsson AS, Sieber BA,
Gigoriou M, Kilkenny C, Salazar-Grueso E, Pachnis V, Arumae U,
Sariola H, Saarma M, Ibanez CF: Functional receptor for GDNF
encoded by the c-RET proto-oncogene. Nature 381:785, 1996
16. Durbec P, Marcos-Gutierrez CV, Kilkenny C, Gigoriou M,
AID
Blood 0037
/
5H33$$$721
Wartiowaara K, Suvanto P, Smith D, Ponder B, Costantini F, Saarma
M, Sariola H, Pachnis V: GDNF signalling through the RET receptor
tyrosine kinase. Nature 381:789, 1996
17. Lin LFH, Doherty DH, Lile JD, Bektesh S, Collins F: GDNF:
A glial cell line-derived neurothropic factor for midbrain dopaminergic neurons. Science 260:1130, 1993
18. Henderson CE, Phillips HS, Pollock RA, Davies AM, Lemeulle C, Armanini M, Simpson LC, Moffet B, Vandlen RA, Koliatsos VE, Rosenthal A: GDNF: A potent survival factor for motoneurons present in the peripheral nerve and muscle. Science 266:1062,
1994
19. Jing S, Wen D, Yu Y, Holst PL, Luo Y, Fang M, Tamir R,
Antonio L, Hu Z, Cupples R, Louis JC, Hu S, Altrock BW, Fox
GM: GDNF-induced activation of the RET protein tyrosine kinase
is mediated by GDNFR-a, a novel receptor for GDNF. Cell 85:1113,
1996
20. Treanor JJS, Goodman L, de Sauvage F, Stone DM, Poulsen
KT, Beck CD, Gray C, Armanini MP, Pollock RA, Hefti F, Phillips
HS, Goddard A, Moore MW, Buj-Bello A, Davies AM, Asai N,
Takahashi M, Vendlen R, Henderson CE, Rosenthal A: Characterization of a multicomponent receptor for GDNF. Nature 382:80, 1996
21. Pachnis V, Mankoo B, Costantini F: Expression of the c-RET
proto-oncogene during mouse embryogenesis. Development 119:
1005, 1993
22. Avantaggiato V, Dathan N, Grieco M, Fabien N, Lazzaro D,
Fusco A, Simeone A, Santoro M: Developmental expression of the
RET proto-oncogene. Cell Growth Diff 5:305,1994
23. Sanchez MP, Silos-Santiago I, Frisen J, He B, Lira SA, Barbacid M: Renal agenesis and the absence of enteric neurons in mice
lacking GDNF. Nature 382:70, 1996
24. Pichel JG, Shen L, Shen HZ, Granholm AC, Drago J, Grinberg A, Lee EJ, Huang SP, Saarma M, Hoffer BJ, Sariola H, Westphal H: Defects in enteric innervation and kidney development in
mice lacking GDNF. Nature 382:73, 1996
25. Moore MW, Klein RD, Farinas I, Sauer H, Armanini M,
Phillips H, Reichardt LF, Ryan AM, Carver-Moore K, Rosenthal A:
Penal and neural abnormalities in mice lacking GDNF. Nature
382:76, 1996
26. Grieco M, Santoro M, Berlingieri MT, Melillo RM, Donghi
R, Bongarzone I, Pierotti MA, Della Porta G, Fusco A, Vecchio G:
PTC is a novel rearranged form of the RET proto-oncogene and is
frequently detected in vivo in human thyroid papillary carcinomas.
Cell 60:557, 1990
27. Flier JS, Underhill LH: The RET proto-oncogene in multiple
endocrine neoplasia type 2 and Hirschprung’s disease. N Engl J Med
335:943, 1996
28. Tahira T, Ishizaka Y, Itoh F, Nakayasu M, Sugimura T, Nagao
M: Expression of the RET proto-oncogene in human neuroblastoma
cell lines and its increase during neuronal differentiation induced by
RETinoic acid. Oncogene 6:2333, 1991
29. Takahashi M, Cooper GM: RET transforming gene encodes
a fusion protein homologous to tyrosine kinases. Mol Cell Biol
7:1378, 1987
30. Takahashi M, Buma Y, Taniguchi, M: Identification of the
RET proto-oncogene products in neuroblastoma and leukemia cells.
Oncogene 6:297, 1991
31. French-American-British (FAB) Cooperative Group: Proposed revised criteria for the classification of acute myeloid leukemia. Ann Intern Med 103:626, 1985
32. The Non-Hodgkin’s Lymphoma Pathologic Classification
Project: National Cancer Institute sponsored study of classifications
of non-Hodgkin’s lymphomas. Summary and description of a Working Formulation for clinical usage. Cancer 42:2112, 1982
33. van Zaanen HCT, Vet RJWM, de Jong CM, von dem Borne
AEGKr, van Oers MHJ: A simple and sensitive method for determin-
03-27-97 13:45:36
blda
WBS: Blood
RET AND GDNFR-a IN HUMAN HEMATOPOIESIS
2937
ing plasma cell isotype and monoclonality in bone marrow using
flow cytometry. Br J Haematol 91:55, 1995
34. Manfioletti G, Gattei V, Buratti E, Rustighi A, De Iuliis A,
Aldinucci D, Goodwin GH, Pinto A: Differential expression of a
novel proline-rich homeobox gene (Prh) in human hematolymphopoietic cells. Blood 85:1237, 1995
35. Carbone A, Gloghini A, Zagonel V, Aldinucci D, Gattei V,
Degan M, Improta S, Sorio R, Monfardini S, Pinto A: The expression
of CD26 and CD40L is mutually exclusive in human T-cell nonHodgkin’s lymphomas/leukemias. Blood 86:4617, 1995
36. Pinto A, Aldinucci D, Gloghini A, Zagonel V, Degan M,
Improta S, Juzbasic S, Todesco M, Perin V, Gattei V, Herrmann F,
Gruus HJ, Carbone A: Human eosinophils express functional CD30
ligand and stimulate proliferation of Hodgkin’s disease cell line.
Blood 88:3299, 1996
37. Gaidano G, Parsa NZ, Tassi V, Della-Latta P, Chaganti RSK,
Knowles DM, Dalla Favera R: In vitro establishment of AIDSrelated lymphoma cell lines: Phenotypic characterization, oncogene
and tumor suppressor gene lesions, and heterogeneity in EpsteinBarr virus infection. Leukemia 7:1621, 1993
38. Testa U, Masciulli R, Tritarelli E, Pastorino R, Mariani G,
Martucci R, Barberi T, Camagna A, Valtieri M, Peschle C: Transforming growth factor- b potentiates vitamin D3-induced terminal
monocytic differentiation of human leukemic cell lines. J Immunol
150:2418, 1993
39. Chomczynsky P, Sacchi N: Single step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction.
Anal Biochem 162:156, 1987
40. Siebert PD, Larrick JW: PCR MIMICs: Competitive DNA
fragments for use as internal standards in quantitative PCR. Biotechniques 14:244, 1993
41. Überla K, Pkatzer C, Diamanstein T, Blankenstein T: Generation of competitor DNA fragments for quantitative PCR. PCR Methods Appl 1:136, 1991
42. Carlomagno E, De Vita G, Berlingieri MT, De Franciscis V,
Melillo RM, Colantuoni V, Kraus MH, Di Fiore PP, Fusco A, Santoro M: Molecular heterogeneity of RET loss of function in Hirsprung’s disease. EMBO J 15:2717, 1996
43. Santoro M, Wong WT, Aroca P, Santos E, Matoskova B,
Grieco M, Fusco A, Di Fiore PP: An epidermal growth factor receptor/RET chimera generates mitogenic and transforming signals: Evidence for a RET-specific signaling pathway. Mol Cell Biol 14:663,
1994
44. Pinto A, Aldinucci D, Gattei V, Zagonel V, Tortora G, Budillon A, Cho-Chung YS: Inhibition of the self-renewal capacity of
blast progenitors from acute myeloblastic leukemia patients by siteselective 8-chloroadenosine 3*,5*-cyclic monophosphate. Proc Natl
Acad Sci USA 89:8884, 1992
45. Pinto A, Attadia V, Fusco A, Ferrara F, Spada OA, Di Fiore
PP: 5-aza-2*-deoxycytidine induces terminal differentiation of leukemic blasts from patients with acute myeloid leukemias. Blood
64:922, 1984
46. Taichman RS, Emerson SG: Human osteosarcoma cell lines
MG-63 and Saoa-2 produce G-CSF and GM-CSF: Identification and
AID
Blood 0037
/
5H33$$$721
partial characterization of cell-associated isoforms. Exp Hematol
24:509, 1996
47. Tahira T, Ishizaka Y, Sugimura T, Nagao M: Expression of
proto-RET mRNA in embryonic and adult rat tissues. Biochem Biophys Res Commun 153:1290, 1988
48. Tsuzuki T, Takahashi M, Asai N, Iwashita T, Matsuyama M,
Asai J: Spatial and temporal expression of the RET proto-oncogene
in embryonic, infant and adult rat tissues. Oncogene 10:191, 1995
49. Yamaguchi Y, Gunji, Nakamura M, Hayakawa K, Maeda M,
Osawa H, Nagayoshi K, Kasahara T, Suda T: Expression of c-kit
mRNA and protein during the differentiation of human hematopoietic progenitor cells. Exp Hematol 21:1233, 1993
50. Rosnet O, Schiff C, Pébusque M-J, Marchetto S, Tonnelle C,
Toiron Y, Birg F, Birnbaum D: Human FLT3/FLK2 gene: cDNA
cloning and expression in hematopoietic cells. Blood 82:1110, 1993
51. Bastard P, Sansilvestri P, Scheinecker C, Knapp W, Debili
N, Vainchenker W, Buring H-J, Monier MN, Kukk E, Partanen J,
Matikainen M-T, Alitalo R, Hatzfeld J, Alitalo K: The tie receptor
tyrosine kinase is expressed by human hematopoietic progenitor cells
and by a subset of megakaryocytic cells. Blood 87:2212, 1996
52. Wasserman R, Li Y-S, Hardy RR: Differential expression of
the BLK and RET tyrosine kinases during B lineage development
is dependent on Ig rearrangement. J Immunol 155:644, 1995
53. Rolink A, Melchers F: Generation and regeneration of cells
of the B-lymphocyte lineage. Curr Opin Immunol 5:207, 1993
54. Satterthwaite A, Witte O: Genetic analysis of tyrosine kinase
function in B cell development. Annu Rev Immunol 14:131, 1996
55. Muroi K, Nakamura M, Amemiya Y, Suda T, Miura Y: Expression of c-kit receptor (CD117) and CD34 in leukemic cells.
Leuk Lymphoma 16:297, 1995
56. Greer JP, Whitlock JA, Mc Kinney: Ki-1 positive anaplastic
large cell lymphoma—A useful concept?, in Armitage J, Newland
A, Keating A, Burnett A (eds): Haematological Oncology, vol 4.
Cambridge Medical Reviews. Cambrige, MA, 1995, p 89
57. Koh HK, Charif M, Weinstock MA: Epidemiology and clinical manifestations of cutaneous T-cell lymphomas. Hematol Oncol
Clin North Am 9:943, 1995
58. Santoro M, Rosati R, Grieco M, Berlingieri MT, ColucciD’Amato L, De Franciscis V, Fusco A: The RET proto-oncogene is
consistently expressed in human pheochromocytomas and thyroid
medullary carcinomas. Oncogene 5:1595, 1990
59. Visser M, Sonneveld RD, Willemze R, Landegent JE: Haematopoietic growth factor tyrosine kinase receptor expression profiles
in normal haematopoiesis. Br J Haematol 94:236, 1996
60. Dickson DW, Mattiace LA, Kure K, Hutchins K, Lyman
WD, Bronsan CF: Microglia in human disease, with an emphasis
on acquired immune deficiency syndrome. Lab Invest 64:135, 1991
61. Trupp M, Ryden M, Jornvall H, Funakoshi H, Timmusk T,
Arenas E, Ibanez CF: Peripheral expression and biological activities
of GDNF, a new neurotrophic factor for avian and mammalian peripheral neurons. J Cell Biol 130:137, 1995
62. Wagner JA: Is IL-6 both a cytokine and a neurothropic factor?
J Exp Med 183:2417, 1996
63. Shrikant P, Benveniste EN: The central nervous system as an
immunocompetent organ. Role of glial cells in antigen presentation.
J Immunol 157:1819, 1996
03-27-97 13:45:36
blda
WBS: Blood