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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3817-3828
Development of a Model for Evaluating the Interaction Between Human
Pre-B Acute Lymphoblastic Leukemic Cells and the Bone Marrow Stromal
Cell Microenvironment
By
Nisha Shah,
LeAnn Oseth, and
Tucker W. LeBien
From the Department of Laboratory Medicine/Pathology, Center for
Immunology, and the Cancer Center, University of Minnesota,
Minneapolis, MN.
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ABSTRACT |
Clonal expansion of B-cell precursor acute lymphoblastic leukemia
(ALL) is potentially regulated by survival, growth, and death signals
transduced by the bone marrow (BM) microenvironment. Using a human BM
stromal cell culture that supports the growth of normal human B-cell
precursors, we established a pre-B ALL cell line designated BLIN-2.
BLIN-2 has a clonal rearrangement of the Ig heavy chain locus, a
dic(9;20) chromosomal abnormality, and a bi-allelic deletion of the
p16INK4a and p19ARF genes. The
most interesting feature of BLIN-2 is an absolute dependence on
adherent human BM stromal cells for sustained survival and growth.
BLIN-2 cultured in the absence of BM stromal cells undergo apoptosis,
and direct contact with viable BM stromal cells is essential for
optimal growth. BLIN-2 cells also grow on vascular cell adhesion
molecule-1 (VCAM-1)-negative human skin fibroblasts, making it
unlikely that a very late antigen-4 (VLA-4)/VCAM-1
interaction is required for BLIN-2 growth. Western blot analysis of
BLIN-2 cells cultured in the presence or absence of BM stromal cells demonstrates that contact of BLIN-2 with BM stromal cells induces hyperphosphorylation of Rb. In contrast, the pre-B ALL cell line BLIN-1, which has a bi-allelic deletion of p16INK4a
p19ARF but does not require BM stromal cells for growth,
does not undergo Rb phosphorylation after BM stromal cell contact. The
BLIN-2 cell line will facilitate identification of ligand/receptor
interactions at the B-cell precursor/BM stromal cell interface and may
provide new insight into microenvironmental regulation of leukemic cell survival and growth.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
ACUTE LYMPHOBLASTIC leukemia (ALL) is a
malignancy characterized by the clonal expansion of T- or B-lineage
lymphoid progenitors, and approximately 70% of newly diagnosed cases
involve CD19+ cells at various stages of B-cell precursor
development.1-3 The cytogenetic and molecular genetic
abnormalities in B-cell precursor ALL frequently consist of chromosomal
translocations involving genes that encode transcription factors, and
many of these transcription factor genes are members of the
homeobox-containing HOX gene family.4,5
Look5 has recently discussed how aberrant regulation of
HOX gene expression, subsequent to the aforementioned translocations, may contribute to the transformation process in ALL.
For example, alterations in HOX gene expression could subvert the normal program of apoptotic fate characteristic of most lymphoid progenitors.5 However, much less understood is the
potential contribution of bone marrow (BM) microenvironmental
survival/growth stimuli to the clonal expansion of leukemic
progenitors, potentially in the background of disrupted apoptotic
programs.
Analyses of various cytokines, interleukins, and colony-stimulating
factors for their capacity to support the survival and growth of B-cell
precursor ALL in vitro have been reported.6-15 Importantly,
several of these studies surveyed a range of cytokines and concluded
that no single cytokine or combination of cytokines could support the
clonogenic growth of leukemic cells from a significant number of
cases.11,14,15 A potential common limitation in all these
studies was a failure to adequately evaluate the clonogenic growth
potential of leukemic blasts using a culture system that recapitulates
the BM microenvironment. B-cell precursor ALL is a malignancy of BM
origin. It follows that at least the earliest stages of B-cell
precursor ALL exhibit a requirement on BM microenvironment-derived survival or growth factor signals for expansion of the leukemic cell
clone. How these BM microenvironment-derived signals influence the
initial stages of transformation or subsequent expansion of the
dominant leukemic subclone is unknown.
A series of studies from Campana et al have described the development
and use of a short-term (ie, 7 days) BM stromal cell-based in vitro
assay for examining the contribution of BM stromal cells to the
survival and programmed cell death of B-cell precursor ALL.16-18 Interestingly, a strong inverse correlation was
observed between the inherent propensity of leukemic blasts from
individual patients to survive on BM stromal cells and the probability
that individual patients would achieve long-term, event-free
survival.18 The probability of event-free survival at
4-year follow-up was lower among patients whose leukemic blasts
survived for up to 7 days on BM stromal cells in vitro compared with
patients whose leukemic blasts underwent apoptosis within 7 days on
stromal cells.
Our laboratory has developed a BM stromal cell culture system that
supports the survival and growth of normal human B-cell precursors.19 The stromal cells in this culture are
predominantly fibroblast-like adventitial reticular cells that express
vascular cell adhesion molecule-1 (VCAM-1).20 Optimal
growth occurs in serum-free medium supplemented with interleukin-7
(IL-7), and direct contact with BM stromal cells is
essential.19,21 In the current study, we describe the
application of this BM stromal cell culture to evaluate the capacity of
BM stromal cells to support the long-term growth of B-cell precursor
ALL. We report the development and characterization of a new B-cell
precursor ALL cell line, designated BLIN-2, that exhibits an absolute
dependency on BM stromal cells for survival and growth. BLIN-2 is a
novel cellular resource for elucidating the ligand/receptor
interactions essential for the development of normal and leukemic human
B-cell precursors.
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MATERIALS AND METHODS |
Establishment of BLIN-2.
Cryopreserved BM from a 3-year-old girl with newly diagnosed B-cell
precursor ALL (based on immunophenotype and histopathology) was thawed
from liquid nitrogen and centrifuged over a Ficoll-Hypaque gradient
(Histopaque; Sigma Chemical Co, St Louis, MO) to remove dead cells.
Approximately 2.0 × 106 recovered interface cells
were washed three times in RPMI-1640 (Life Technologies, Grand Island,
NY), supplemented with 2% fetal bovine serum (FBS; Hyclone, Ogden,
UT), and plated onto a pre-established adherent layer of adult BM
stromal cells in 96-well flat-bottom microtiter plates (Costar,
Cambridge, MA). One objective at this stage was to determine whether
leukemic B-cell precursors would exhibit optimal growth when cultured
on BM stromal cells supplemented with IL-7, as we had previously shown
for normal human pro-B cells.19,21 Leukemic cells were
therefore cultured in the absence or presence of IL-7 (10 ng/mL;
PeproTech, Rocky Hill, NJ) at an initial density of 3.5 × 104 cells/well. After 3 weeks in culture,
approximately 50% of the wells had viable leukemic cells as judged by
inverted light microscopy, independent of whether the leukemic cells
were cultured in the presence or absence of IL-7. The cellular content
of wells with viable cells was diluted 1:2 and passaged onto fresh
adult BM stromal cells. After an additional 3 weeks, the leukemic cells were pooled from multiple wells and phenotyped. The leukemic cells established in culture at this stage were designated B lineage-2 (BLIN-2). Because IL-7 had no additional effect on BLIN-2 survival or
growth beyond the supportive effect of the stromal cells, IL-7 supplementation was discontinued.
Other cells.
BLIN-1 is a surface µ+/ light chain+ pre-B
ALL cell line originally established in this laboratory.22
RAMOS is an Epstein-Barr virus (EBV)-negative, surface
µ+/surface  light chain+ Burkitt lymphoma
cell line obtained from the American Type Culture Collection (ATCC;
Rockville, MD). BLIN-1 and RAMOS were maintained in RPMI-1640
supplemented with 10% FBS, 100 U penicillin/mL, and 100 µg
streptomycin/mL. Fluorescence-activated cell sorting (FACS)-purified fetal BM pro-B cells were isolated as previously
described.21
Adherent cells.
Fetal BM was obtained from 19- to 21-week gestational age fetuses, in
accordance with guidelines set forth by the University of Minnesota
Committee on the Use of Human Subjects in Research. A detailed
description of the methods we use for the establishment of human BM
stromal cells has been published.23,24 Briefly, total BM
mononuclear cells were isolated by Ficoll-Hypaque centrifugation and
seeded into 75-cm2 tissue culture flasks (Falcon, Lincoln
Park, NJ) in RPMI-1640 containing 10% FBS. Nonadherent cells were
washed off after 2 hours at 37°C, and the adherent cells were
cultured in EX-CELL 610 (JRH Biosciences, Lenexa, KS) supplemented with
10% FBS, 100 U/mL penicillin, and 100 µg/mL of streptomycin. An
adherent layer of BM stromal cells was generally established within 1 week. The cells were then detached with 0.05% trypsin, 0.53 mmol/L
EDTA (Life Technologies, Grand Island, NY) and transferred into new 75-cm2 flasks containing fresh EX-CELL 610/10% FBS. The
adherent layer reached confluence within 1 week, and the cells were
passaged a second time. Adult BM stromal cells were established and
passaged in the same manner as fetal BM stromal cells, except that
adult BM stromal cells required 2 to 3 more days to reach confluence. Upon reaching 90% confluence after second passage, fetal and adult BM
stromal cells could be maintained in EX-CELL 610 serum-free medium.
Foreskin fibroblasts were originally obtained at first passage from Dr
Elizabeth Wayner (formerly of the Department of Laboratory Medicine/Pathology, University of Minnesota, Minneapolis, MN). These
cells were maintained and passaged as described above for BM stromal
cells and were used between passages 8 and 11 in the experiments
described herein.
Antibodies.
Monoclonal antibodies (MoAbs) recognizing specific Ig molecules or
membrane proteins and their conjugation to fluorescein isothiocyanate
(FITC) or biotin have been
described.19-21,23,24 Streptavidin-phycoerythrin (PE) was
purchased from Caltag Laboratories (South San Francisco, CA). Goat
antimouse FITC was purchased from Southern Biotechnology Associates,
Inc (Birmingham, AL). Rabbit antibody recognizing the human
retinoblastoma (Rb) protein was purchased from Santa Cruz Biotechnology
(Santa Cruz, CA) as a horseradish peroxidase conjugate (catalog no.
SC-050-HRP) and was used in Western blotting. Rabbit anti-Rb was made
against a peptide corresponding to amino acids 914-928 at the carboxy terminus of p110 Rb and recognizes phosphorylated and nonphosphorylated forms of Rb.
Immunofluorescent staining and flow cytometry.
These methods have been previously described.19-21,23
Cytogenetics and fluorescence in situ hybridization (FISH).
Metaphase cells were harvested from short-term unstimulated cultures of
the patient's BM or from the actively growing BLIN-2 cell line and
were subsequently G-banded using Wright stain according to standard
cytogenetic procedures.25 In the initial analysis of
BLIN-2, 15 metaphase cells were analyzed microscopically for definition
of the clonal abnormalities. FISH was performed using chromosome 9 and
chromosome 20 whole chromosome paint probes (VYSIS Inc, Downersgrove,
IL) directly labeled in Spectrum orange and Spectrum green,
respectively. FISH was performed with modification of the
manufacturer's instructions. Briefly, target DNA was denatured, hybridized with the probes for 16 hours, and washed in 2× SSC at
72°C. Chromosomes were counterstained with 4 ,
6-diamidino-2-phenylindole (DAPI), and metaphase cells
and hybridization signals were visualized under an Olympus
microscope outfitted for fluorescence with a triple band
pass (B-MAX) filter (Olympus Optical Co Ltd, Tokyo, Japan).
Growth assay.
Adherent cells were seeded into 96-well flat-bottom microtiter plates
at 0.6 to 1.0 × 104 cells/well in EX-CELL 610/10%
FBS. After 3 to 5 days, the adherent cells were washed with X-VIVO
10/0% FBS and BLIN-2 cells were seeded at 1 to 3 × 104 cells/well in a final volume of 200 µL of X-VIVO
10/0% FBS. X-VIVO 10 is a serum-free medium purchased from
BioWhittaker, Inc (Walkersville, MD). It contains human serum albumin
as a carrier protein and insulin and transferrin as growth-promoting
supplements. The capacity of BLIN-2 cells to grow in the absence of BM
stromal cell contact was tested by plating BLIN-2 cells into 6.5-mm
transwells (Costar) containing a 0.4-µm polycarbonate membrane
suspended above the adherent cells. The cultures were fed every 3 to 4 days by replacing 50% of the culture volume with fresh X-VIVO 10/0%
FBS, unless otherwise stated. BLIN-2 growth was quantified using
PE-conjugated anti-CD19 in a microsphere flow cytometric-based
quantitation assay.21
Apoptosis and cell cycle analysis.
Cells were cultured at 1 × 106 cells/60-mm petri
plate in 4 mL of X-VIVO 10 serum-free medium in the presence or absence
of fetal BM stromal cells. At each time point, BLIN-2 cells were gently
removed from culture without disrupting the adherent layer and
simultaneously analyzed for subdiploid events and cell cycle using the
method of Nicoletti et al.26 Briefly, the cell pellet was
gently resuspended in 1 mL of hypotonic solution (50 µg/mL propidium
iodide in 0.1% sodium citrate plus 0.1% Triton-X-100) and incubated
overnight at 4°C in the dark. Analysis of intact and fragmented
nuclei was achieved using a FACSCalibur flow cytometer (Becton
Dickinson and Co, Mountain View, CA) and CELLQuest software. Chromatin
degradation, a characteristic of apoptosis, was detected as a
heterogeneous subdiploid population to the left of the peak corresponding to intact nuclei in G0/G1 phases of the cell cycle.
FITC-conjugated Annexin V was purchased from Pharmingen (San Diego, CA)
and used according to the manufacturer's instructions. Briefly, cells
were washed once in ice-cold phosphate-buffered saline (PBS) and then
washed once in prewarmed binding buffer (10 mmol/L HEPES, pH 7.4, 140 mmol/L NaCl, 2.5 mmol/L CaCl2). Cells were resuspended in
100 µL binding buffer containing 5 µL of Annexin V-FITC and 10 µL
of propidium iodide (50 µg/mL of stock solution) and then incubated
at room temperature for 15 minutes. An additional 400 µL of binding
buffer was added to the cells before analysis using a FACSCalibur flow
cytometer and CELLQuest software (Becton Dickinson, San Jose, CA).
Polymerase chain reaction (PCR) and Southern blotting.
TRI Reagent (Molecular Research Center, Cincinnati, OH) was used to
extract DNA from BLIN-1, RAMOS, and BLIN-2 cells. Approximately 0.5 µg of DNA was amplified using 0.4 mmol/L of each specific primer, 0.4 mmol/L of dNTP, 1.5 mmol/L MgCl2, and 2.5 U AmpliTaq (Perkin Elmer, Branchburg, NJ) in a final volume of 50 µL. The mixture was overlaid with 100 µL mineral oil. The PCR reaction was
performed in an automated DNA Thermal Cycler (Hybaid Ltd, Middlesex,
UK) with the following parameters: denaturation at 95°C for 4 minutes, 30 cycles of 94°C for 1 minute, annealing at 55°C for
1 minute, 1 minute of extension at 72°C, and a final 10 minutes of
extension at 70°C. Amplified products were electrophoresed on 1.5%
agarose gels. The gels were washed in a denaturing solution containing
0.5N NaOH and 1.5 mol/L NaCl for 20 minutes at room temperature,
followed by one wash in a neutralization solution containing 1.5 mol/L
NaCl and 0.5 mol/L Tris-HCl, pH 7.5, for 30 minutes at room
temperature. The DNA was then transferred onto a nylon membrane
(Nytran; Schleicher & Schuell, Keene, NH) using vacuum blotting with a
PosiBlot (Stratagene, La Jolla, CA) and the membrane was UV
cross-linked with 120 mJ/cm2 (UV Crosslinker; Fisher
Scientific, Pittsburgh, PA) and allowed to air-dry. Probes were
end-labeled with ATP[ -32P] using T4 Polynucleotide
Kinase (Life Technologies) according to the manufacturer's
recommendations. Blots were prehybridized in 1 mol/L NaCl, 0.2 mol/L
Tris-HCl, pH 7.5, 0.1% sodium dodecyl sulfate (SDS), containing 200 µg/mL denatured salmon sperm for 2 hours at 49°C to 51°C.
Hybridization was conducted in the same solution and at the same
temperature as the prehybridization, with the addition of
ATP[-32P]-labeled probes for 18 hours. The membranes
were washed sequentially with 1× SSC, 0.1% SDS twice at room
temperature for 10 minutes each and then at 54°C to 58°C for 10 minutes, followed by a final wash at room temperature for 5 minutes.
The membrane was developed by autoradiography using X-Omat AR film
(Eastman Kodak, Rochester, NY) at  70°C. Exposure times
ranged from 3 to 6 hours.
The primer pairs used were as follows: p15INK4b
exon 2, 5-CGA-GGA-GAA-CAA-GGG-CAT-3 and
5-GAA-TGC-ACA-CCT-CGC-CAA-CG-3;
p19ARF exon 1 ,
5-AGT-CTG-CAG-TTA-AGG-GGG-CAG-3 and
5-GGC-TAG-AGA-CGA-ATT-ATC-TGT-3; p16INK4a exon 1 ,
5-GAG-GCG-GCG-AGA-ACA-TGG-TG-3 and
5-CTT-CTA-GGA-AGC-GGC-TGC-TG-3; p16INK4a-p19ARF exon 2, 5-GCT-TCC-TTT-CCG-TCA-TGC-CG-3 and
5-CAA-ATT-CTC-AGA-TCA-TCA-GTC-C-3; actin,
5-ATC-ATG-TTT-GAG-ACC-TTC-AA-3 and
5-CAT-CTC-TTG-CTC-GAA-GTC-CA-3. The sequence of the
internal probes used to detect specific amplified products by Southern
blotting were as follows: p15INK4b exon 2, 5-CAA-ATC-TAC-ATC-GCG-ATC-TAG-G-3;
p19ARF exon 1,
5-CAC-CAA-ACA-AAA-CAA-GTG-CG-3;
p16INK4a exon 1,
5-GAC-GCT-GGC-TCC-TCA-GTA-GC-3;
p16INK4a-p19ARF exon 2, 5-CTG-TTC-TCT-CTG-GCA-GGT-CAT-G-3; and actin,
3-ATG-TCA-CGC-ACG-ATT-TCC-CG-5.
Western blotting.
BLIN-1 or BLIN-2 cells were cultured on BM stromal cells or in medium
alone for 18 hours. The leukemic cells were gently poured off the BM
stromal cell adherent layer, lysed in 0.05 mol/L Tris, 0.25 mol/L NaCl,
0.5% NP-40, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 1%
aprotinin, 10 µg/mL leupeptin, 0.1 mol/L NaF, and 0.002 mol/L sodium
orthovanadate and then vortexed for 30 minutes at 4°C. The lysates
were centrifuged for 30 minutes at 13,500g at 4°C. The
supernatant was removed and protein quantitation was conducted using
the Coomassie Plus Protein Assay Reagent (Pierce, Rockford, IL). Forty
to 50 µg of protein per sample was electrophoresed on a 10%
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel and then
transferred onto a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). The membrane was blocked in Tris-buffered saline-Tween (TBST; 50 mmol/L Tris, pH 7.6, 150 mmol/L NaCl, 0.1% Tween 20) containing 5% milk for 2 hours at room temperature. Horseradish peroxidase-conjugated rabbit anti-Rb was incubated with the blot at a
final concentration of 2 µg/mL for 30 minutes in TBST containing 5%
milk. The membrane was then washed 4 times at 10-minute intervals in
TBST and developed by enhanced chemiluminescence according to the
manufacturer's instructions (Amersham Life Science, Arlington Heights,
IL).
Quantitation of chemiluminescent Western blots was conducted by
scanning densitometry using a model GS-700 Imaging Densitometer (Bio-Rad, Hercules, CA). Data analysis was conducted using Molecular Analyst ver 2.0 software. The intensity of individual
bands was converted to a histogram profile of the sum of the pixel
density, and the profiles were adjusted to remove background. The area under the curve was calculated in square millimeters and calibrated to
an internal machine constant. Band intensity values were expressed as
ratios of hyperphosphorylated Rb to the total Rb in each lane. Scanning
densitometry was conducted using the facilities of the Biomedical
Imaging and Processing Laboratory, University of Minnesota Medical
School.
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RESULTS |
Establishment of the BLIN-2 cell line.
This study was initiated to establish BM stromal cell-dependent pre-B
ALL cell lines as part of a long-term project to elucidate how BM
stromal cells influence the development of this malignancy. Twenty-three consecutive BM specimens from patients with newly diagnosed ALL were plated on allogeneic BM stromal cell feeders in
X-VIVO 10 serum-free medium in the presence or absence of IL-7. Of
these 23 specimens, 17 showed a less than 10-fold increase in
CD19+ cell number after 4 weeks in culture, whether IL-7
was present or not. Six specimens showed a greater than 10-fold
expansion in CD19+ cell number. One of these cultures,
designated BLIN-2, is the subject of this report.
The BLIN-2 cell line was initiated in April 1993 using cryopreserved BM
from a pediatric patient diagnosed with ALL. The cryopreserved leukemic
BM specimen was 100% CD19+ and 50% CD34+ at
the time of initial plating onto adult BM stromal cells, and a stable
population of leukemic cells emerged after approximately 6 weeks. As
shown in Fig 1, the established cell line
expressed a typical pre-B phenotype (ie, CD19+,
CD20+, CD22+, CD34 , weakly
surface µ+, light chain+, and  /
light chain). Southern blot analysis of DNA isolated
from the original cryopreserved leukemic BM specimen and BLIN-2 cells
in culture for 6 months showed identical heavy and light chain Ig gene
rearrangements (data not shown). DNA from the original cryopreserved
leukemic BM specimen and BLIN-2 was amplified by PCR using consensus
VH family primers and consensus primers that would amplify
JH1, JH3, and JH4 genes. The
amplified products were sequenced, and the original cryopreserved
leukemic BM specimen and the established BLIN-2 cell line were shown to
harbor identical VH3(DN4)JH4
rearrangements across 80 nucleotides of sequence (including identical N
region insertions). This confirmed the clonal identity of the original BM leukemic cells and the established BLIN-2 cell line using a unique
V(D)J rearrangement as a fingerprint.
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| Fig 1.
Immunophenotype of BLIN-2. Background staining using
isotype-matched myeloma proteins as negative controls is shown by the
horizontal bar in the upper left part of each histogram.
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G-banded chromosomal analysis of the patient's BM at diagnosis in June
1993 showed a hypodiploid clone with a 45,XX complement, including an
abnormal chromosome 9 with a deletion of its distal short arm and loss
of one chromosome 20 as the sole karyotypic abnormalities. G-banded
chromosomal analysis of the BLIN-2 cell line in June 1994 showed these
same abnormalities and also a gain of a chromosome 8. The origin of the
trisomy 8 in BLIN-2 cells is unknown. It may have been present as a
rare subclone in the original BM specimen that expanded in culture, or
it could have arisen during evolution of the cell line in culture.
In February 1998, G-banded chromosomal analysis of the BLIN-2 cell line
was repeated and supplemented by FISH using chromosome 9 and chromosome
20 paint probes (Fig 2). FISH showed that
the abnormal chromosome 9 was actually a dicentric chromosome
containing the short arm of a chromosome 20 joined to the long arm of a
chromosome 9, with centromeric regions of both chromosomes included in
the rearrangement. Thus, the karyotype of this cell line has been stable in culture and can be definitively designated as
46,XX,+8,dic(9;20)(p11;q11.1). This karyotype results in net losses of
one copy each of the short arm of chromosome 9 and the long arm of
chromosome 20 and a net gain of one copy of chromosome 8.
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| Fig 2.
FISH analysis of BLIN-2 cells from February 1998. Normal
chromosome 9 is shown in red, normal chromosome 20 in green, and
dic(9;20) as red/green. Chromosomes were counterstained with DAPI
(blue).
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Stromal cell dependence of BLIN-2.
The BLIN-2 cell line was established on an adherent monolayer of BM
stromal cells and has maintained a strict BM stromal cell requirement
for survival and growth. BLIN-2 growth showed a good correlation with
the number of BM stromal cells on which BLIN-2 was initially plated. As
shown in Fig 3, when BLIN-2 cells were plated on 100% confluent BM stromal cells, there was an eightfold increase in BLIN-2 by day 14. In contrast, 10% confluent BM stromal cells only supported a 3.5-fold increase in BLIN-2 by day 14, and 1%
confluent BM stromal cells or serum-free medium alone failed to support
survival or growth. We emphasize that this BM stromal cell culture
system does not require serum supplementation. Addition of up to 20%
vol/vol FBS does not enhance growth of BLIN-2 on BM stromal cells.
Furthermore, BLIN-2 cells cultured in the absence of BM stromal cells
do not grow in X-VIVO 10 serum-free medium supplemented with FBS.
Growth of BLIN-2 is also not contingent upon a specific source of human
BM stromal cells, because we have continuously maintained BLIN-2 cells
on BM stromal cells established from greater than 20 different donors.
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| Fig 3.
Growth of BLIN-2 on BM stromal cells. BLIN-2 cells (2.5 × 104/well) were cultured on various concentrations of BM
stromal cells, and the number of BLIN-2 cells was quantified on days 7, 10, and 14. Each symbol represents the mean ± SD of triplicate wells.
One hundred percent confluency corresponds to approximately 6 × 103 BM stromal cells per 200-µL flat-bottom microtiter
well. This experiment is representative of five similar experiments.
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The role of direct BM stromal cell contact in supporting BLIN-2 growth
was evaluated using a transwell system.
Figure 4 compared the growth
characteristics of an early passage of BLIN-2 cryopreserved in February
1995 and thawed in January 1998 for use in this experiment versus
BLIN-2 cells that had been passaged continuously. Figure 4A and B shows
that direct contact with BM stromal cells was essential for optimal
BLIN-2 growth. The February, 1995 BLIN-2 cells cultured in transwell
inserts slowly died over the 15-day culture period and increased
10-fold in contact with BM stromal cells (Fig 4A). In contrast, the
continuously passaged BLIN-2 cells underwent a very modest fourfold
increase by day 15; this compared with the 33-fold increase under
contact conditions by day 15. When BLIN-2 cells were cultured in direct
contact with BM stromal cells, and a transwell containing BLIN-2 cells
was inserted above the BLIN-2/BM stromal cell contact culture, BLIN-2
cells in the transwell failed to grow, whereas BLIN-2 cells in direct
contact with BM stromal cells grew normally (data not shown). These
results make it unlikely that BLIN-2 cell contact induced the BM
stromal cells to secrete a product that supported BLIN-2 growth.
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| Fig 4.
The role of BM stromal cell contact in the growth of
BLIN-2. BLIN-2 cells cryopreserved in February 1995 and thawed for use
in this experiment in January 1998 (A) or maintained continuously on BM
stromal cells (B) were cultured in direct contact with BM stromal
cells, in transwell inserts (noncontact) suspended above the BM stromal
cells, or in medium alone. BLIN-2 growth was quantified on days 7, 11, or 15. Each symbol represents the mean ± SD of triplicate wells. This
experiment is representative of six similar experiments.
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The importance of BM stromal cell integrity was tested by assaying the
capacity of BLIN-2 cells to grow on Triton X-100-lysed or
paraformaldehyde-fixed BM stromal cells. As shown in
Fig 5, fixed or lysed BM stromal cells were
unable to support BLIN-2 survival and growth. Thus, intact,
metabolically active BM stromal cells are essential for BLIN-2 growth.
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| Fig 5.
Growth of BLIN-2 on fixed or lysed BM stromal cells. BM
stromal cells were fixed in 1% paraformaldehyde or lysed in 1% Triton
X-100 and then washed five times in medium before the addition of
BLIN-2 cells. BLIN-2 growth was quantified on days 4 and 8. Each bar
represents the mean ± SD of triplicate wells. This experiment is
representative of four similar experiments.
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The growth kinetics of BLIN-2 on BM stromal cells (Figs 3, 4, and 5 and
numerous other experiments) indicated that the population doubling time
of BLIN-2 is relatively slow. BLIN-2 would typically undergo a twofold
to threefold increase in cell number between days 0 and 7 and a
fivefold to sevenfold increase in cell number between days 7 and 14. As
presented in Fig 6A, BLIN-2 cells
repetitively passaged on BM stromal cells showed a consistent sixfold
to eightfold increase in cell number during the initial 14 days after
transfer onto fresh stromal cells. This rate of population increase was similar to IL-7-stimulated normal human pro-B cells cultured on BM
stromal cells (Fig 6B).
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| Fig 6.
Comparison of BLIN-2 and normal pro-B cell growth on BM
stromal cells. BLIN-2 and FACS-purified normal pro-B cells were plated
at 2.5 to 5.0 × 104/well on BM stromal cells and cultured
in X-VIVO 10/0% FBS. The pro-B cells were supplemented with 10 ng/mL
of IL-7. BLIN-2 and pro-B cell numbers were quantified on day 7 and
replated onto fresh BM stromal cells (transfer 1) at the initial cell
concentration of 2.5 to 5.0 × 104/well. Quantitation of
cell numbers and replating onto fresh BM stromal cells was repeated on
day 14 (transfer 2) and day 21 (transfer 3).
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Apoptosis of BLIN-2 cells in the absence of BM stromal cells.
BLIN-2 cells were cultured on BM stromal cells or in serum-free medium
alone, and propidium iodide-stained nuclei and nuclear fragments were
analyzed by flow cytometry at various time points. Figure 7 shows that BLIN-2 cells cultured
on BM stromal cells did not exhibit an increase in subdiploid events
during 4 days of culture. In contrast, BLIN-2 cells cultured in
serum-free medium alone showed no accumulation of subdiploid events at
day 1, but underwent an increase to approximately 27% subdiploid
events at day 2, that reached approximately 60% by day 4. In a
separate experiment shown in Fig 8, Annexin
V binding was coupled with propidium iodide staining to quantify early
stage apoptotic events (ie, Annexin V+, propidium
iodide). BLIN-2 cells cultured on BM stromal cells
for 4 days (Fig 8A) had 2% Annexin V+/propidium
iodide apoptotic events and less than 10% Annexin
V+/propidium iodide+ events that represent dead
cells. In contrast, BLIN-2 cells cultured in medium alone for 4 days
(Fig 8B) had 5.7% Annexin V+/propidium
iodide apoptotic events and greater than 60%
Annexin V+/propidium iodide+ dead cells.
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| Fig 7.
Flow cytometric analysis of subdiploid events in BLIN-2
cells cultured in the presence or absence of BM stromal cells.
BLIN-2/stromal cell cultures were gently shaken to displace BLIN-2 from
the stromal cells. These stromal cell-displaced BLIN-2 cells and BLIN-2
cells in medium alone were then lysed in 0.1% Triton X-100, and the
nuclei were isolated and stained with propidium iodide. The percentage
of subdiploid events is listed in each histogram. This experiment is
representative of six similar experiments.
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| Fig 8.
Annexin V/propidium iodide dual staining of BLIN-2 cells
cultured in the presence (A) or absence (B) of BM stromal cells for 4 days. BLIN-2/stromal cell cultures were gently shaken to displace
BLIN-2 from the stromal cells. These stromal cell-displaced BLIN-2
cells and BLIN-2 cells in medium alone were then stained with propidium
iodide and FITC-conjugated Annexin V. The numbers represent the
percentage of propidium iodide+/Annexin V+
or propidium iodide/Annexin V+ cells as a
function of total cells analyzed.
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BLIN-2 growth does not depend on very late antigen-4
(VLA-4)/VCAM-1 interaction.
BM stromal cells used in our culture system are fibroblast-like
adventitial reticular cells that express VCAM-1.20,23
Figure 9 shows that human foreskin
fibroblasts were as effective as BM stromal cells in supporting BLIN-2
growth, even though we could not detect VCAM-1 on the surface of
foreskin fibroblasts by flow cytometry (data not shown). Furthermore,
inclusion of saturating concentrations of MoAb recognizing VCAM-1, or
the 4 or 1 subunits of VLA-4, had no effect on BM stromal
cell-dependent growth when used individually or in combination (data
not shown). Thus, VCAM-1 binding to VLA-4 and signaling pathways
activated after VLA-4 cross-linking are probably not essential for
BLIN-2 growth. BM stromal cells and foreskin fibroblasts were
comparable in their capacity to support BLIN-2 growth. However, human
umbilical vein endothelial cells, the murine S17 BM stromal cell line,
and NIH-3T3 fibroblasts did not support BLIN-2 growth (data not shown).
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| Fig 9.
Growth of BLIN-2 on VCAM-1+ BM stromal
cells or VCAM-1 foreskin fibroblasts. BLIN-2 growth was
quantified on days 7, 14, and 19. Each symbol represents the mean ± SD of triplicate wells. This experiment is representative of eight
similar experiments.
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Potential role of the retinoblastoma (Rb) pathway in BLIN-2 growth.
Homozygous deletions or point mutations in the
p16INK4a cyclin-dependent kinase (CDK) inhibitor
gene on chromosome 9p are found in many freshly isolated human tumors
and tumor cell lines, including ALL.27,28 Furthermore, the
p16INK4a locus in mouse29 and
human30,31 includes a novel gene, designated p19ARF (for alternative reading frame), that uses
an exon (exon 1) that is 13 to 20 kb centromeric to exon 1 used
by p16INK4a. Because BLIN-2 has a dic(9;20), we
evaluated the status of the p16INK4a,
p19ARF, and p15INK4b genes by
PCR. To reduce the possibility that analysis of BLIN-2 DNA would be
confounded by contaminating BM stromal cell DNA, we FACS-purified
BLIN-2 before conducting the PCR. As shown in Fig 10, BLIN-2 has deleted both alleles
of p19ARF exon 1, p16INK4a
exon 1, and p16INK4a-p19ARF
exon 2. However, PCR amplification using p15INK4b
exon 2-specific primers indicated that at least one
p15INK4b allele was present. These results contrast
with RAMOS cells that contain an intact INK4a locus and the
BLIN-1 pre-B ALL cell line that has the three genes deleted.
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| Fig 10.
Status of INK4 locus genes in BLIN-2. DNA was
isolated from RAMOS, BLIN-1, and BLIN-2 using TRI reagent and amplified
with primers specific for various INK4 locus exons, as
described in Materials and Methods. BLIN-2 was separated from BM
stromal cells by FACS sorting before DNA isolation. The RAMOS Burkitt
lymphoma cell line served as a positive control for amplification of
all INK4 locus genes.
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With the results in Fig 10 showing that both
p16INK4a alleles are deleted in BLIN-2, we
hypothesized that growth of BLIN-2 on BM stromal cells may occur by
virtue of continuous activation of the cyclin D/CDK/Rb pathway. As an
initial approach in testing this hypothesis, we examined the status of
Rb phosphorylation in BLIN-2 and BLIN-1 cells in the presence or
absence of BM stromal cell contact. The pre-B ALL cell line BLIN-1 was
used as a control, because it has both p16INK4a
alleles deleted (Fig 10) but is not dependent on BM stromal cells for
growth. As shown in Fig 11, two isoforms
of Rb were present in BLIN-2 cells cultured in the presence or absence
of BM stromal cells for 18 hours, but a difference in the relative
ratio of hypo-phosphorylated (pRb) to hyper-phosphorylated (ppRb) Rb
was detected. Scanning densitometry indicated that 80% of Rb was
hyperphosphorylated in BLIN-2 cells cultured on BM stromal cells (Fig
11, lane 4), whereas only 47% of Rb was hyperphosphorylated in BLIN-2
cells maintained in medium alone (Fig 11, lane 3). In contrast, the
control BLIN-1 cell line exhibited only one predominant
hyper-phosphorylated Rb isoform, independent of whether the cells were
maintained on BM stromal cells or in medium alone.
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| Fig 11.
Phosphorylation of Rb in BLIN-2 cells cultured in the
presence or absence of BM stromal cells. BLIN-1 or BLIN-2 cells were
cultured in X-VIVO 10 serum-free medium for 18 hours in the absence
() or presence (+) of BM stromal cells. The cells were then
harvested and lysed in 0.5% NP-40, and approximately 50 µg of
protein per lane was electrophoresed on a 10% SDS-PAGE gel. The
separated proteins were then transferred to nitrocellulose and Western
blotted with rabbit antihuman Rb. The blot was restained with mouse
antihuman tubulin to control for equal protein loading. The numbers
under each lane represent the percentage of Rb detected as the
hyper-phosphorylated isoform (ppRb  pRb + ppRb) by scanning
densitometry.
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The kinetics of Rb phosphorylation in BLIN-2 was evaluated by
time-course analysis. BLIN-2 cells were cultured in medium alone for 18 hours, replated onto fresh BM stromal cells, and assessed for
subsequent changes in Rb phosphorylation. As expected, BLIN-2 cells
cultured in medium alone for 18 hours expressed more
hypo-phosphorylated Rb (Fig 12, lane 4),
compared with BLIN-2 cells maintained on BM stromal cells for 18 hours
(Fig 12, lane 3). The pattern of Rb phosphorylation in BLIN-1 was the
same whether the cells were cultured on BM stromal cells (Fig 12, lane
1) or in medium alone (Fig 12, lane 2). These results are consistent
with the results in Fig 11, except that, in the experiment shown in Fig
12, three distinct phosphorylated Rb isoforms are visible. When BLIN-2
cells cultured in medium alone for 18 hours (Fig 12, lane 4) were
transferred to fresh BM stromal cells, a shift toward
hyper-phosphorylated Rb was detected between 6 and 9 hours. The absence
of detectable Rb in BM stromal cells provided a useful control to
confirm that BLIN-2 cell protein lysates were not contaminated with BM
stromal cell proteins.
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| Fig 12.
Time course of Rb phosphorylation in BLIN-2. Lanes 1 through 4, BLIN-1 and BLIN-2 were cultured for 18 hours and analyzed
for Rb expression/phosphorylation as described in the Fig 10 legend.
Lanes 5 through 9, BLIN-2 cells from lane 4 were replated on fresh BM
stromal cells and reanalyzed at the times indicated for Rb
expression/phosphorylation. The blot was restained with mouse antihuman
tubulin to control for equal protein loading.
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DISCUSSION |
This report describes the establishment and characterization of a novel
human pre-B ALL cell line with an absolute requirement on BM stromal
cells or skin fibroblasts for optimal growth. A large number of ALL
cell lines have been generated from patients with B-cell precursor or
T-lineage ALL. The vast majority of these cell lines proliferate in
FBS-supplemented tissue culture medium in the absence of any adherent
feeder layer (Zhang et al32 and references therein),
although one study did present a brief description of a B-cell
precursor ALL cell line that required BM stromal cells for
growth.33 We have previously reported that BM stromal
cell-dependent human B-cell development can occur in the absence of
IL-7,34 consistent with the fact that SCID patients with
mutations in the c subunit of the IL-7 receptor or Jak-3 tyrosine
kinase have normal (or even elevated) numbers of B
cells.35-38 Although some B-cell precursor ALL respond to
IL-7, IL-7 by itself is not a potent growth stimulus for the majority
of B-cell precursor ALL.11-15 Thus, BM stromal cell
products in addition to IL-7 must be essential for survival
and/or growth of normal and leukemic human B-cell precursors.
We therefore sought to establish ALL cell lines that would preserve a
dependence on BM stromal cells for long-term survival and growth in
vitro to establish a model to evaluate the role of the BM
microenvironment in the development of B-cell precursor ALL.
BLIN-2 has a typical pre-B cell phenotype, including the expression of
cell surface µ heavy chains, light chains, CD19, CD20, and CD22
(Fig 1). Data in Figs 3, 4, and 5 demonstrate that (1) viable, intact
BM stromal cells are essential for BLIN-2 growth and (2) optimal growth
requires direct contact. Although removal of BLIN-2 from BM stromal
cells leads to inevitable apoptotic cell death, more recent passages of
BLIN-2 exhibit slightly greater survival in noncontact (transwell)
conditions (Fig 4A and B). Furthermore, earlier passages of BLIN-2
doubled every 4 to 5 days, whereas recent passages double approximately
every 2 days (Fig 4A and B). These data suggest that selection of a
subclone (or subclones) with greater proliferative capacity has
occurred during continuous passage of BLIN-2 on BM stromal cells. The
genetic differences that underlie this modest change in survival and
proliferation status are unknown. The population doubling time of
BLIN-2 on BM stromal cells is comparable to the population doubling
time of normal pro-B cells stimulated with IL-7 (Fig 6). One
interpretation of these data would argue that BLIN-2 and normal pro-B
cells are dependent on similar BM stromal cell products for survival
and growth. The difference in long-term growth characteristics (ie, permanent growth of BLIN-2 vis-à-vis limited growth of normal pro-B cells) could then be at least partially attributed to the absence
of p16INK4a and/or p19ARF proteins in
BLIN-2 cells, leading to dysregulation in G1 to S-phase entry
and/or entry into apoptosis (see below). However, normal pro-B
cells plated on BM stromal cells supplemented with IL-7 undergo partial
differentiation to the pre-B and immature B stages of B-cell
development.21 Thus, the ability of normal pro-B cells to
differentiate and the inability of BLIN-2 to differentiate may also
explain the results.
BLIN-2 cells removed from BM stromal cells gradually undergo apoptosis
(Figs 7 and 8), consistent with the relatively slow growth rate of this
cell line. BLIN-2 cells cultured in the absence of BM stromal cells
exhibited a sharp increase in subdiploid events between days 1 and 2 that reached 60% by day 4 (Fig 7). However, analysis of the early
stages of apoptosis by Annexin V binding to exposed phosphatidylserine
residues indicated only a minor percentage (5.6%; Fig 8B) of Annexin
V+/propidium iodide events 4 days after
the removal of BLIN-2 cells from BM stromal cells. The difference
between the results in Figs 7 and 8 probably reflects the difference in
the two assays. The Annexin V binding assay detects early stages of
apoptosis in intact cells.39 In contrast, propidium iodide
analysis of subdiploid events essentially detects nuclear fragments,
which do not necessarily correlate with the number of apoptotic
cells.40
A major question not resolved in the current study is the identity of
the BM stromal cell-derived ligand (or ligands), and their cognate
receptors on BLIN-2, essential for the survival and growth of BLIN-2.
The data in Fig 4 indicate that direct contact with BM stromal cells is
essential for optimal growth, but minimal survival/growth also occurs
in noncontact conditions. We have tested a variety of cytokines and
combinations of cytokines for their capacity to support survival/growth
of BLIN-2 in the absence of BM stromal cells. Log range concentrations
of IL-3, IL-6, IL-7, IL-9, IL-10, IL-11, stem cell factor, Flt3 ligand,
basic fibroblast growth factor, or various combinations failed to
enhance the survival of BLIN-2 cells beyond that which occurred in
medium alone (N. Shah and T.W. LeBien, unpublished
observations). A potential candidate in the contact
equation would be VLA-4/VCAM-1, because BLIN-2 cells express the
CD49d/4 subunit of VLA-4 (Fig 1) and the BM stromal cells used in
our culture system express VCAM-1.20 Studies using mouse
and human early B-lineage cells have suggested a role for VLA-4/VCAM-1
in the growth and development of B-cell precursors.41,42 Furthermore, chimeric mice derived by injecting embryonic stem cells
containing a targeted deletion of the 4 gene into RAG-1- or
RAG-2-deficient blastocysts exhibited a dramatic deficiency of
4/ B cells in adult BM, blood, and
spleen.43 However, other signaling interactions between
B-lineage cells and BM stromal cells are not dependent on a
VLA-4/VCAM-1 adhesion event.44,45 Independent of the
precise role of VLA-4/VCAM-1 in other in vitro or in vivo experimental
systems, VLA-4/VCAM-1 interaction plays little or no role in the
survival and growth of BLIN-2. This conclusion is supported by two
separate results. First, BLIN-2 cells show essentially identical growth
rates on VCAM-1+ BM stromal cells and VCAM-1
foreskin fibroblasts (Fig 9). Second, inclusion of saturating concentrations of MoAb against the 4 or 1 subunits of VLA-4 and/or MoAb against VCAM-1 has no effect on the growth of
BLIN-2 on BM stromal cells (N. Shah and T.W. LeBien, unpublished
observations).
As shown in Fig 2, BLIN-2 harbors a dic(9;20), a recurring chromosomal
abnormality previously identified in a small subset of
children46,47 and adults48 with pre-B ALL.
Whether a direct relationship exists between the presence of a
dic(9;20) and the BM stromal cell requirements of this cell line is
currently unknown. Additional cell lines containing a dic(9;20) would
need to be established and studied to address this question. Given that
the breakpoints involved in the formation of the dic(9;20) occur within heterochromatin regions, it is highly unlikely that a chimeric gene is
created at the point of fusion. More likely, the significant ramification of this rearrangement is the resulting monosomy for 9p and
20q. Of particular relevance may be the loss of the entire INK4a locus mapped to 9p21.
The INK4a locus on chromosome 9p21 includes the cell cycle
inhibitor genes p15INK4b,
p16INK4a, and
p19ARF.27-31 As shown in Fig 10, BLIN-2
has both alleles of p16INK4a and
p19ARF deleted. The PCR results do not allow us to
determine whether one or both alleles of p15INK4b
are intact. However, all of the chromosomal DNA telomeric to 9p11 has
been deleted on one copy of chromosome 9, making it likely that only
one copy of p15INK4b is present in BLIN-2 cells.
Chromosomal 9p21 rearrangements or deletions in ALL can result in the
loss of both alleles of p16INK4a, but preserve one
or both alleles of p15INK4b.49-51
Although less is known about the disposition of
p19ARF in ALL, Gardie et al52 recently
reported an evaluation of this gene in 87 cases of T-lineage ALL. These
investigators found that p19ARF was deleted or
disrupted in 75 cases of T-lineage ALL harboring recombination events
in the INK4a locus; yet, in 4 of the 75 cases, p16INK4a was not deleted or altered. On the basis
of these data, they proposed that p19ARF may be
targeted by the genetic events that occur at the INK4 locus in
T-lineage ALL. To our knowledge, a comparable analysis of
p16INK4a and p19ARF in B-cell
precursor ALL has not been reported.
Two recent reports have demonstrated that
p16INK4a-deficient leukemic cell lines
reconstituted with wild-type p16INK4a un |
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Abstract
Introduction