Download Stromal cells regulate survival of B-lineage leukemic

From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
NEOPLASIA
Stromal cells regulate survival of B-lineage leukemic cells during chemotherapy
Ryan E. Mudry, James E. Fortney, Teresa York, Brett M. Hall, and Laura F. Gibson
Approximately 20% of B-lineage acute
lymphoblastic leukemias are not cured by
traditional chemotherapy. The possibility
was examined that residual leukemic cells
that potentially contribute to relapse are
harbored in association with fibroblastic
stromal cells in the bone marrow. Modulation of cytarabine (Ara-C) and etoposide
(VP-16) efficacy by bone marrow stromal
cells in vitro was investigated. Stromal
cell coculture was shown to sustain the
proliferation of B-lineage leukemic cells
and to reduce leukemic cell apoptosis
when exposed to Ara-C or VP-16. Direct
contact with stromal cells was essential
for the protection of leukemic cells during
chemotherapy, whereas soluble factors
had negligible effect. Specifically, signaling mediated through interaction with the
stromal cell adhesion molecule VCAM-1
was required to maintain the maximum
viability of leukemic cells during Ara-C
and VP-16 exposure. In contrast, the interaction of leukemic cells with fibronectin
did not confer significant resistance to
either chemotherapeutic agent. These observations suggest a role for the bone
marrow microenvironment in modulating
the response of B-lineage leukemic cells
to Ara-C or VP-16, and they indicate specific molecular interactions that may be
important in determining the sensitivity
of leukemic cells to treatment. (Blood.
2000;96:1926-1932)
© 2000 by The American Society of Hematology
Introduction
Acute lymphoblastic leukemia (ALL) is a disease that initiates and
progresses in the bone marrow. The marrow microenvironment is
presumed to play an essential regulatory role in the development of
this cancer. In addition, the bone marrow microenvironment
provides a primary site in which residual leukemic cells may
survive standard chemotherapy.1,2 After treatment, surviving cells
contribute to relapse and to poor prognosis of patients for whom
ALL is not successfully eradicated by traditional chemotherapy.
The role of bone marrow stromal cells in modulating the response
of leukemic cells to chemotherapy has not been previously
investigated.
Stromal cells influence the development of hematopoietic cells
through the production of cytokines and through the signals
mediated by direct contact of progenitor cells with stromal cells.
The stromal cell adhesion molecules VCAM-1 and fibronectin
have been identified as 2 of the critical molecules necessary for the
adhesion of progenitors to stromal cells.3-6 Many of the developmental cues provided by stromal cells to normal hematopoietic
progenitor cells have also been shown to impact the survival and
expansion of leukemic cells. Like their normal counterparts, many
B-lineage ALL cells express VLA-4, which can interact with
VCAM-1 and fibronectin on the surfaces of stromal cells.7 Primary
ALL cells isolated from patients maintain the greatest viability
when in direct coculture with an adherent stromal cell layer, and
this effect is not duplicated by stromal cell-conditioned medium.8
The optimal survival of leukemic cells through direct interaction
with stromal cells indicates signals mediated through adhesion that
cannot be completely substituted by soluble factors.
In the current study, we investigated bone marrow stromal cell
modulation of Ara-C– and VP-16–induced cell cycle arrest and
apoptosis of the ALL cell lines JM-1, SUP-B15, and RS4.
Coculture of B-lineage leukemic cells with stromal cells diminished the effect of both chemotherapeutic agents on cell cycle
progression and apoptosis. This effect could be attributed to the
interaction of leukemic cells with VCAM-1 but not with fibronectin. These observations suggest a role for bone marrow stromal
cells in maintaining leukemic cell division and survival during
exposure to chemotherapy, and they are relevant to treatment
strategies aimed at minimizing ALL relapse through the reduction
of residual leukemic cells in bone marrow.
Materials and methods
Leukemia and bone marrow stromal cell cultures
Growth factor independent cell lines derived from patients with B-lineage
acute lymphoblastic leukemia/lymphoma used in this study were obtained
from the ATCC (Manassas, VA). The panel of cell lines chosen represented
the diversity of phenotypes that characterize common and pre-B ALL. They
included JM-1 (#CRL-10423; HLA DR⫹, CD10⫹, CD19⫹, surface Ig ⫺
VLA-4⫹, VLA-5⫺), RS4 (#CRL-1873; HLA DR⫹, CD9⫹, CD24⫹, VLA4⫹, VLA-5⫹ cytoplasmic and surface Ig⫺), and SUP-B15 (#CRL-1929;
HLA DR⫹ CD5⫹, CD10⫹, CD13⫹ CD38⫹, CD71⫹, cytoplasmic Ig⫹, Ph⫹,
VLA-4⫹, VLA-5⫹). In addition to the expression of the antigens listed
above, RS4 cells contained the (4;11)(q21;23) translocation frequently
present in infants younger than 1 year of age with ALL. An additional
T-lineage leukemic cell line, Jurkat (ATCC #TIB-152, CD3⫹, VLA-4⫹,
VLA-5⫹), was included in viability experiments to evaluate lineage
specificity of stromal cell effects. All leukemic cell lines were grown in
From the Departments of Pediatrics, Microbiology and Immunology, and
Biology, and the Blood and Marrow Transplantation Program, Mary Babb
Randolph Cancer Center, West Virginia University, Morgantown, WV.
West Virginia University, P. O. Box 9214, Morgantown, WV 26506; e-mail:
[email protected].
Submitted February 23, 2000; accepted May 4, 2000.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported in part by the Elsa U. Pardee Foundation (L.F.G.), and a West
Virginia University Research Development Grant (L.F.G.).
Reprints: Laura F. Gibson, Department of Pediatrics, Health Sciences Center,
1926
© 2000 by The American Society of Hematology
BLOOD, 1 SEPTEMBER 2000 䡠 VOLUME 96, NUMBER 5
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
BLOOD, 1 SEPTEMBER 2000 䡠 VOLUME 96, NUMBER 5
STROMAL CELL REGULATION OF APOPTOSIS
medium with supplements, as recommended by the ATCC. The establishment of human bone marrow stromal cells by our laboratory has been
previously described.9
1927
included JM-1 cells in medium alone, JM-1 cells in 5 ␮mol/L VP-16 or 1.0
␮g/mL Ara-C, with all groups cocultured with fixed and control stromal
cells. After 48 and 72 hours of culture, JM-1 cells were collected, and
viability of triplicate samples was evaluated.
Chemotherapeutic agents
Ara-C (Sigma, St Louis, MO) was stored at ⫺20 °C at 10 ␮g/␮L and VP-16
(Bristol Laboratories, Princeton, NJ) at a stock concentration of 1 g/50 mL.
Both drugs were diluted in Iscove medium immediately before use.
Experimental doses of Ara-C (.01-1.0 ␮g/mL) and VP-16 (1.0-5.0 ␮mol/L)
were used to approximate doses described for use in regimens for acute
lymphoblastic leukemia.10
Cell cycle analysis
To evaluate the cell cycle profile of B-lineage leukemic cells in the presence
of stromal cells, JM-1 cells were collected after treatment with 5 ␮mol/L
VP-16 or 1.0 ␮g/mL Ara-C for 24 to 48 hours in the presence or absence of
an adherent stromal cell layer. JM-1 cells were fixed in EtOH, treated for 30
minutes with RNase (Sigma), and stained with propidium iodide for
DNA analysis.
Evaluation of leukemic cell viability
B-lineage JM-1, RS4, and SUP-B15 or T-lineage Jurkat cells were cultured
in medium alone or were cocultured with an adherent layer of bone marrow
stromal cells for 24 to 72 hours in the presence or absence of VP-16 (1-5
␮mol/L) or Ara-C (0.01-0.1 ␮g/mL). Leukemic cells were removed from
adherent stromal cells by vigorous pipetting. Viability was evaluated by
trypan blue exclusion in triplicate samples.
Annexin staining
After 48 and 72 hours of culture in the presence of 1 to 5 ␮mol/L VP-16 or
0.1 to 1.0 ␮g/mL Ara-C, JM-1 cells were collected and stained using an
Annexin V–fluorescein isothiocyanate (FITC) kit (Genzyme, Cambridge,
MA) according to the recommended protocol of the manufacturer to detect
apoptotic cells. All treatment groups were established in the presence and
absence of an adherent stromal cell layer. As a representative cell line, JM-1
cells were collected on a Becton Dickinson Flow Cytometer, and data
analyzed using CellQuest software and cytometer (Becton Dickinson,
Mountainview, CA).
Evaluation of JM-1 viability in stromal cell conditioned medium
Conditioned medium was collected from confluent cultures of stromal cells
after 72 hours of incubation. JM-1 cells were subsequently cultured in 75%
conditioned medium for 24 to 72 hours in the presence or absence of VP-16
(1 and 5 ␮mol/L) or Ara-C (0.01 and 0.1 ␮g/mL). Viability was evaluated
by trypan blue exclusion in triplicate samples.
Glutaraldehyde fixation of stromal cells
To interrupt the metabolic activity of stromal cells while leaving surface
proteins intact, confluent stromal cell layers were treated with glutaraldehyde as previously described.11,12 Briefly, confluent stromal cells were
treated with 2% glutaraldehyde (FisherChemical, Fair Lawn, NJ) for 5
minutes and rinsed 5 times with fresh medium. Stromal cells were then
incubated for 12 hours in medium, rinsed, and incubated an additional 12
hours in fresh medium before the addition of JM-1 cells. Treatment groups
Culture of JM-1 and RS4 cells on fibronectin-coated plates
To determine whether interactions with the stromal cell adhesion molecule
fibronectin altered chemotherapy-induced apoptosis in leukemic cells,
JM-1 (VLA-4⫹, VLA-5 ⫺) or RS4 (VLA-4⫹, VLA-5⫹) cells were incubated
on fibronectin-coated 24-well plates (Sigma) for up to 72 hours in the
presence of 0.1 to 1.0 ␮g/mL Ara-C, 1 to 5 ␮mol/L VP-16, or medium
alone. Matched samples were cultured in uncoated 24-well plates. Cells
were collected by vigorous pipetting, and all samples were evaluated in
triplicate by trypan blue exclusion.
Blocking VCAM-1 on bone marrow stromal cells
To determine whether the observed protective effect of stromal cells
resulted from VCAM-1 binding, JM-1 or RS4 cells were incubated on
confluent stromal cells treated with mouse antihuman VCAM-1 antibody
(Santa Cruz Biotechnology, Santa Cruz, CA) during Ara-C and VP-16
treatment. Before the addition of leukemic cells and drugs, stromal cell
layers were pretreated with 5 ␮g of a blocking anti–VCAM-1 monoclonal
antibody for 30 minutes at room temperature. At 24-hour intervals, 2.5
␮g/mL fresh antibody was added to cocultures. Viability of leukemic cells
cultured on anti–VCAM-1–treated stromal cells was compared to leukemic
cells on control stromal cells and to stromal cells treated with an identical
concentration of isotype-matched control antibody. In addition, JM-1 and
RS4 cell viability in medium, in the presence and absence of drug, was
evaluated. At the termination of culture, leukemic cells were collected, and
viability was evaluated in triplicate by trypan blue exclusion.
Results
Bone marrow stromal cells impact JM-1 cell cycle kinetics
during chemotherapy exposure
As a representative B-lineage leukemic cell line, JM-1 cell cycle
status was evaluated after treatment with VP-16 or Ara-C. Data
shown represent cell cycle profiles of JM-1 cells after 24 hours of
exposure to each chemotherapeutic agent (Table 1). After treatment
with Ara-C or VP-16, JM-1 cells accumulated in G1 of the cell
cycle. The presence of stromal cells (SC) in culture abrogated this
G1 block. JM-1 cells treated with Ara-C in medium alone (NS) had
73% of cells in Go/G1, compared with 42% in Go/G1 when treated
in the presence of stromal cells. JM-1 cells treated with VP-16 for
24 hours had 83% in Go/G1 compared to 58% in Go/G1 when treated
in the presence of stromal cells. Consistent with this observation,
stromal cell coculture maintained a higher proportion of JM-1 cells
in S phase during treatment than cells exposed to chemotherapy in
medium alone (Table 1). Untreated cultures had fewer cells in
Go/G1 when stromal cells were present than in cultures that did not
contain stromal cells. Higher numbers of JM-1 cells in the S and
G2/M phases of the cell cycle were also recorded when JM-1 cells
Table 1. Stromal cell coculture maintains JM-1 cell cycle progression
JM-1 cells recovered
(⫻105/mL)
%S
% G0/G1
% G2/M
Treatment
NS
SC
NS
SC
NS
SC
NS
Control
SC
12.2
15.1
56.6
45.3
34.4
44.9
9.0
9.8
Ara-C
8.9
14.3
73.2
42.0
24.0
45.9
2.8
12.1
VP-16
10.0
13.7
83.2
58.0
14.7
37.7
2.1
4.3
JM-1 cells were collected after treatment with 5 ␮mol/L VP-16 or 0.1 ␮g/mL Ara-C for 24 hours in the presence (SC) or absence (NS) of an adherent stromal cell layer. JM-1
cells were fixed in EtOH, treated for 30 minutes with RNase, and stained with propidium iodide for DNA analysis. Data are representative of 2 independent experiments.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1928
MUDRY et al
BLOOD, 1 SEPTEMBER 2000 䡠 VOLUME 96, NUMBER 5
were cultured with stromal cells than in control cultures that did not
contain stromal cells. Absolute numbers of JM-1 cells were
enumerated in all cultures. Stromal cell coculture modestly increased the absolute number of JM-1 cells recovered after treatment (Table1). In addition, after chemotherapy, JM-1 cells cocultured with stromal cells were collected and moved to new adherent
layers, which confirmed their ability to repopulate cultures (data
not shown).
Coculture of B-lineage leukemic cells with bone marrow
stromal cells decreases the effectiveness of VP-16 and Ara-C
To determine whether coculture with a stromal cell underlayer affected
leukemic cell survival during chemotherapy exposure, 3 B-lineage
leukemic cells lines and T-lineage Jurkat cells were grown on stromal
cells and compared to cells maintained in medium alone. In all cases,
stromal cell coculture resulted in increased JM-1, RS4, and SUP-B15
cell viability during treatment (Figure 1). After 72 hours of treatment
with 0.1 ␮g/mL Ara-C, JM-1 cell viability was reduced to 16% in
medium alone compared to 79% when cocultured with bone marrow
stromal cells (Figure 1A). JM-1 cells treated with 5 ␮mol/L VP-16 in
medium were 35.3% viable, compared to 53.8% when treated with
stromal cells (Figure 1B). Untreated leukemic cells cultured on stromal
cells remained approximately 100% viable and were not different from
controls maintained in medium alone. Protection by stromal cell
coculture was also observed when RS4 or SUP-B15 cultures were
evaluated at 48 hours (Figure 1C). Stromal cell coculture consistently
resulted in higher viability of B-lineage leukemic cells after treatment.
RS4 cells treated with VP-16 in medium were 10.7% viable, compared
to 23.6% when treated in the presence of stromal cells. VP-16 treatment
of SUP-B15 cells reduced viability to 3.4% in medium alone, compared
to 25.1% when treated during stromal cell coculture (Figure 1C).
Comparable trends were noted at 24 and 72 hours (data not shown).
Stromal cell protection was also observed during Ara-C treatment
(Figure 1C). In contrast, Jurkat cell viability after treatment with Ara-C
or VP-16 was not improved by stromal cells (Figure 1C). Similar
viability data were recorded when leukemic cells were removed by
vigorous pipetting or trypsinization.
Apoptosis is reduced in leukemic cells treated with
chemotherapy in the presence of an adherent stromal cell layer
To determine whether VP-16 and Ara-C induced apoptotic cell
death, JM-1 cells were evaluated by annexin staining after chemotherapy exposure in the presence or absence of bone marrow
stromal cells. The presence of stromal cells resulted in reduced
numbers of apoptotic JM-1 cells in cultures treated with VP-16 or
Ara-C (Figure 2).
Soluble stromal cell-derived factors do not enhance leukemic
cell survival during VP-16 or Ara-C treatment
To determine whether soluble factors derived from stromal cells
altered the response of leukemic cells to chemotherapy, JM-1 cells
were cultured in stromal cell-conditioned medium in the presence
or absence of VP-16 or Ara-C as described, and viability evaluated.
No significant differences in JM-1 viability were observed during
Ara-C or VP-16 treatment in conditioned medium compared to
control fresh medium (Figure 3).
Interaction with stromal cell surface proteins altered the
response of JM-1 cells to Ara-C and VP-16
Stromal cells were fixed in 2% glutaraldehyde and used in
coculture experiments as described above. Viability of JM-1 cells
Figure 1. B-lineage leukemic cell viability is enhanced during Ara-C or VP-16
treatment by bone marrow stromal cells. (A) JM-1 cells were cultured in medium
alone or with an adherent layer of bone marrow stromal cells for 24 to 72 hours in the
presence or absence of Ara-C or (B) VP-16 at indicated concentrations. Leukemic cells
were removed by vigorous pipetting, and viability was evaluated by trypan blue exclusion in
triplicate samples. Data shown are representative of 4 independent experiments. (C) RS4,
SUP B-15, or Jurkat cells were cultured for 48 hours in medium containing 0.1 ␮g/mLAra-C
(solid black bars) or 1 ␮mol/L VP-16 (open bars). Leukemic cells were treated with the
same concentration of Ara-C (hatched bars) or VP-16 (light gray bars) during coculture with
an adherent layer of bone marrow stromal. Leukemic cells were removed by vigorous
pipetting, and viability was evaluated as above. Data shown are mean ⫾ SEM and are
representative of 2 independent experiments.
treated on control or fixed stroma was significantly higher than
medium alone in all groups (Tukey test; P ⫽ ⬍ .001). Coculture
with untreated stromal cells increased the viability of JM-1 cells
exposed to VP-16 from 16% to 30% after 48 hours and from 9% to
24% after 72 hours. Glutaraldehyde-fixed stromal cells increased
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
BLOOD, 1 SEPTEMBER 2000 䡠 VOLUME 96, NUMBER 5
STROMAL CELL REGULATION OF APOPTOSIS
1929
Figure 2. Chemotherapy-induced apoptosis of JM-1 cells is delayed by coculture with stromal cells. After 48 and 72 hours of culture in 0.1 ␮g/mL Ara-C (A, B) or
5 ␮mol/L VP-16 (C, D), JM-1 cells were collected as described and stained with
Annexin-V–FITC. Samples included untreated JM-1 control cells (control), JM-1 cells
treated on an adherent stromal cell layer (stroma), and JM-1 cells treated in medium
alone (no stroma). JM-1 cells were collected on a Becton Dickinson Flow Cytometer,
and data were analyzed using CellQuest software.
the viability of VP-16–treated JM-1 cells to 34% and 26% at 48 and
72 hours, respectively. JM-1 viability during treatment with Ara-C
was increased comparably by coculture with control or fixed
stromal cells (Figure 4). Adherence of JM-1 cells to stromal cells
was not affected by fixation (data not shown).
Interaction with fibronectin did not enhance survival of JM-1
or RS4 cells during chemotherapy exposure
To determine whether interaction with the stromal cell adhesion
molecule fibronectin conferred part of the protection against
chemotherapy-induced apoptosis in JM-1 or RS4 cells, leukemic
cells were incubated on fibronectin-coated plates in the presence of
Ara-C, VP-16, or medium alone as previously described. Incubation of JM-1 cells on fibronectin-coated plates during 24 hours or
Figure 3. Stromal cell-conditioned medium does not significantly alter the
response of JM-1 cells to Ara-C or VP-16. JM-1 cells were cultured in 75% stromal
cell conditioned medium for 24 to 72 hours in the presence or absence of VP-16 or
Ara-C, as described in “Materials and methods.” Viability was evaluated by trypan
blue exclusion in triplicate samples. Data shown are mean ⫾ SEM and are
representative of 3 independent experiments.
Figure 4. Glutaraldehyde fixation of bone marrow stromal cells does not alter
chemotherapy protection. Confluent stromal cells were treated with 2% glutaraldehyde for 5 minutes and were thoroughly rinsed before the addition of JM-1 cells.
Matched control stromal cells were cultured in medium alone. JM-1 cells cultured on
control or glutaraldehyde-treated stromal cells were exposed to 5 ␮mol/L VP-16 or
1.0 ␮g/mL Ara-C. Chemotherapy-treated JM-1 cells were also evaluated in medium
alone. After 48 and 72 hours of culture, JM-1 cells were collected, and viability of
triplicate samples were evaluated by trypan blue exclusion.
48 hours of Ara-C and VP-16 exposure did not protect JM-1 cells
from chemotherapy-induced cell death (Figure 5).
Signaling through adhesion of JM-1 or RS4 cells to VCAM-1
was required for maximal protection from Ara-C– and
VP-16–induced cell death
To evaluate the contribution of VCAM-1 in adhesion in protection
from chemotherapy, binding was interrupted using a blocking
anti–VCAM-1 antibody. After culture in 0.1 ␮g/mL Ara-C or 5
␮mol/L VP-16 as previously described, viability of JM-1 or RS4
cells was significantly lower in cultures containing stromal cells
treated with anti–VCAM-1 antibody than in untreated stromal cells
(Figure 6). After 48 hours of treatment with Ara-C, JM-1 cells in
medium alone had viability reduced to approximately 8% from a
control value of 96%. The presence of stromal cells increased
Ara-C– or VP-16–treated JM-1 viability to approximately 23%.
Treatment of stromal cells with anti–VCAM-1 antibody abrogated
the protective effect of stromal cell coculture. Treatment of stromal
cell layers with a matched isotype control antibody did not
diminish the protective capacity of stromal cells in any sample
(data not shown).
Similarly, incubation of RS4 cells with anti–VCAM-1–treated bone
marrow stromal cells diminished the protective capacity of stromal cells
during Ara-C and VP-16 treatment (Figure 7B). After 72 hours of
treatment in medium alone, RS4 viability was reduced to 17% in Ara-C
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1930
MUDRY et al
BLOOD, 1 SEPTEMBER 2000 䡠 VOLUME 96, NUMBER 5
be attributed to the expression of VCAM-1 on the stromal cell surface.
This observation has significant implications for the design of new
chemotherapeutic regimens to treat this disease.
VLA-4 expression on leukemic cells of lymphoid and myeloid
origin has been shown to facilitate adhesion to bone marrow
stromal cells through binding to VCAM-1 in vitro.4,15,16
Pre-incubation of an adherent cell layer with anti–VCAM-1
antibody significantly blocked the adhesion of pre-B ALL cells
derived from 19 patients.17 This report was consistent with
observations of normal immature B-cell precursors that were
shown to have reduced adhesion to marrow-derived fibroblasts
when treated with antibodies to the ␣ and ␤ chains of VLA-4.18
Functional relevance of these observations was suggested by an in
vivo model in which a variant pre-B ALL cell line, which lacked
VLA-4 expression, had reduced ability to establish leukemia in
sublethally irradiated SCID mice.19 In this study, mice with
established leukemia had significantly less marrow infiltration with
Figure 5. Adhesion to fibronectin does not protect JM-1 cells from Ara-C– or
VP-16–induced cell death. JM-1 cells were incubated in 24-well plates for 24 or 48
hours in the presence of 1.0 ␮g/mL Ara-C, 5 ␮mol/L VP-16, or medium alone
(control). Culture plates used were coated with fibronectin (black bars) or uncoated
(gray bars). JM-1 cells were collected by vigorous pipetting and evaluated in triplicate
by trypan blue exclusion. A representative graph of 3 independent experiments is
presented. Data shown are mean ⫾ SEM.
and 2% in VP-16 treated cultures. Stromal cell coculture during
treatment increased the viability of RS4 cells exposed to either
chemotherapeutic drug. Consistent with JM-1 cells, incubation of
stromal cells with anti–VCAM-1 antibody reduced the viability of RS4
on stromal cells from 38% to 23% in cultures containing Ara-C and
from 30% to 11% in cultures exposed to VP-16 (Figure 7B). JM-1 and
RS4 cells were shown to lack VCAM-1 expression by flow cytometric
analysis (data not shown).
Discussion
Recognition that microenvironmental cues impact tumor cell response
to treatment is not unique to leukemic cells. Previous studies have
investigated the responses of various tumor cell types to chemotherapy
and reported an association of responses with metastatic site. In one
report, the response of colon carcinoma to doxorubicin and 5-flurouracil
was correlated with 3 distinct anatomic sites: lung, liver, and spleen.13 In
vivo sites that conferred maximal doxorubicin resistance had elevated
levels of protein kinase-C activity in the carcinoma cells tested.14 These
observations suggest that signaling pathways may be impacted by
factors that are unique to specific sites in the microenvironment. In the
current report, we describe the effect of bone marrow stromal cells on
survival of 3 B-lineage leukemic cell lines. The presence of an intact
stromal cell layer conferred dramatic protection from chemotherapyinduced apoptotic cell death. This protective effect of stromal cells could
Figure 6. Disruption of VCAM-1 binding abrogates stromal cell protection of
JM-1 cells during chemotherapy. JM-1 cells were incubated on confluent stromal
cells treated with mouse antihuman VCAM-1 antibody during Ara-C and VP-16
treatment. Before the addition of leukemic cells and chemotherapeutic drugs, stromal
cell layers were pretreated with 5 ␮g anti–VCAM-1; 2.5 ␮g/mL fresh antibody was
added at 24-hour intervals, as described in “Materials and methods.” Viability of
leukemic cells cultured on anti–VCAM-1–treated stromal cells was compared to that
of JM-1 cells on control stromal cells and to that of stromal cells treated with an
isotype-matched control antibody. In addition, JM-1 cells in medium, in the presence
and absence of drug, were included. After 48 to 72 hours of culture, JM-1 cells were
collected and evaluated in triplicate by trypan blue exclusion. Data shown are
mean ⫾ SEM and are representative of 3 independent experiments.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
BLOOD, 1 SEPTEMBER 2000 䡠 VOLUME 96, NUMBER 5
Figure 7. Fibronectin does not protect VLA-4ⴙ VLA-5ⴙ RS4 cells from Ara-C or
VP-16–induced cell death. (A) RS4 cells were incubated in 24-well plates for 72 hours in
the presence of 0.1 ␮g/mL Ara-C, 5 ␮mol/L VP-16, or medium alone (control). Culture
plates used were coated with fibronectin (black bars) or uncoated (gray bars). RS-4 cells
were collected by vigorous pipetting and were evaluated in triplicate by trypan blue
exclusion. (B) RS-4 cells were incubated on confluent stromal cells treated with mouse
antihuman VCAM-1 antibody during Ara-C and VP-16 treatment. Before the addition of
leukemic cells and chemotherapeutic drugs, stromal cell layers were pretreated with 5 ␮g
anti–VCAM-1; 2.5 ␮g/mL fresh antibody was added at 24-hour intervals, as described in
“Materials and methods.” Viability of leukemic cells cultured on anti–VCAM-1–treated
stromal cells was compared to RS-4 cells on control stromal cells. Data shown are mean ⫾
SEM and are representative of 2 independent experiments.
the VLA-4⫺ variant of NALM-6.19 Consistent with in vivo data
from this study, VLA-4⫺ cells had reduced binding to, and
migration beneath, an adherent murine stromal cell layer.19 These
reports emphasize the role of VLA-4 interactions with VCAM-1 in
mediating the adhesion of leukemic cells to stromal cells in the
marrow and support the usefulness of in vitro coculture models in
which stromal and leukemic cell interactions can be studied.
The importance of understanding interactions between leukemic
progenitor cells and the bone marrow microenvironment is based not
only on the marrow being the site in which disease is initiated, but also
on the finding that bone marrow is the site of first relapse for
approximately 60% to 70% of childhood ALL.1,2 If the initial relapse is
at an extramedullary site, such as the central nervous system, marrow
disease has often developed and is detectable by PCR.1 In a study of 13
children with ALL relapse outside the bone marrow, disease was
detected in marrow samples of 12 children.1 In vivo models have
suggested a negative correlation between the number of residual
leukemic cells in the marrow and disease-free survival,20 making
interactions between leukemic cells and bone marrow stromal cells
important to investigate.
In the current study it was noted that the cell cycle progression
of JM-1 cells is maintained when stromal cells are present in
STROMAL CELL REGULATION OF APOPTOSIS
1931
cultures during chemotherapy exposure, in contrast to disrupted
cycling of leukemic cells exposed to chemotherapy in the absence
of stromal cells (Table 1). Modestly increased numbers of JM-1
cells were recovered from cultures after treatment during coculture
(Table 1). Regulation of the expression or activity of proteins that
control cell cycle progression is one way stromal cells may
influence malignant cells associated with it in vivo. Signals that
alter intracellular phosphorylation are a potentially rapid and
effective method by which stromal cells modulate production or
activity of leukemic cell proteins. A recent report suggested Rb
phosphorylation as one factor impacted by stromal cell coculture of
a leukemic cell line BLIN-2.21 Hyperphosphorylation of Rb
subsequent to exposure to stromal cells may contribute to dysregulated cell cycle progression and may be one mechanism by which
stromal cells augment leukemic cell proliferation.
In addition to maintaining proliferation during treatment, stromal cell coculture delayed the onset of apoptosis in JM-1 and
enhanced viability of JM-1, RS4, and SUP-B15 cells (Figures 1 and
2). We previously described the regulation of bcl-2 and bax in
normal murine pro-B cells by contact with bone marrow stromal
cells.22 Based on similarities between B-lineage ALL cells and
normal B-cell progenitors, it is likely that the stromal cell
microenvironment may impact expression of these apoptotic
regulatory molecules in leukemic cells as well. Interestingly,
elevated bcl-2 expression has been shown to block chemotherapyinduced apoptosis of the human pre-B leukemic cell line, 697, in
response to a wide variety of agents, including etoposide (VP-16)
and 1-␤-D-arabinofuranosyl-cytosine (Ara-C).23,24 Chronic lymphocytic leukemia cells (CLL) have also been shown to have glucocorticoid and chemotherapy sensitivity affected by the endogenous
ratio of BCL-2:BAX.25 A recent report indicated that the human
B-lymphoma cells line, JLP119, altered BAX conformational
changes during etoposide treatment if stimulated with VCAM-1 or
IL-4, mediators that can be found in the bone marrow microenvironment.26 These observations emphasize stromal cell influence of
apoptotic regulatory molecules as a likely mechanism by which the
microenvironment modulates response to chemotherapy.
In the current study, stromal cell-conditioned medium conferred
negligible protection to leukemic cells during VP-16 and Ara-C
exposure (Figure 3). Therefore, the role of proteins on the stromal cell
surface was investigated with glutaraldehyde-fixed stromal cells (Figure
4). Previous reports have shown this fixation process to disrupt
adequately the metabolism of adherent 3T3 cells, allowing investigation
of the role of signaling through ligand–receptor interactions in the
absence of cytokine production.11 It has been reported that signaling
between stromal cells and hematopoietic progenitors can be bidirectional, with the adhesion of hematopoietic cells to stromal cells altering
stromal cell function.27 This presented the possibility that the adhesion
of JM-1 cells to stromal cells resulted in alterations of stromal cell
function that were protective during chemotherapy. However, in our
study, adhesion to fixed stromal cells resulted in viability of chemotherapy-treated JM-1 cells that was not different from viable stromal
cells (Figure 4). In addition, we investigated the possibility that stromal
cell adherent layers may sequester chemotherapeutic agents, with
diminished leukemic cell death during coculture resulting from reduced
drug concentrations in the culture medium. However, comparisons of
Ara-C and VP-16 activity in medium cocultured on stromal cells layers
versus medium alone indicated no significant change in effectiveness of
drug (data not shown). JM-1 cells were killed as efficiently by Ara-C or
VP-16 pre-incubated for 48 hours on a confluent stromal cell layer as
they were by the same concentration of drug incubated for 48 hours in
medium alone (data not shown). These observations support the
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1932
BLOOD, 1 SEPTEMBER 2000 䡠 VOLUME 96, NUMBER 5
MUDRY et al
conclusion that signals that protect leukemic cell survival primarily
result from cell surface interactions.
It is interesting that the interaction of JM-1 cells with surface
VCAM-1 was protective during Ara-C and VP-16 exposure
(Figures 6 and 7B) but that fibronectin was not (Figures 5, 7A).
Both VCAM-1 and fibronectin bind to VLA-4 on JM-1 cells;
however, these ligands bind VLA-4 at different sites on the
molecule.28 These data suggest that VCAM-1 binding results in
distinctly different intracellular signals in JM-1 cells than results
from fibronectin binding. Alternatively, the activation state of
VLA-4 might not have been such that optimal binding to, or
signaling through, VLA-4 occurred. To determine whether the
presence of VLA-5, in addition to VLA-4, on the leukemic cell
surface supported protection through interaction with fibronectin,
the response of RS4 cells expressing both receptors was investigated. Again, interaction with fibronectin did not significantly
protect from Ara-C– or VP-16–induced cell death (Figure 7A). This
observation further supports the importance of signaling that
occurs downstream of interaction with VCAM-1. Interestingly,
Jurkat cells that express both VLA-4 and VLA-5 did not benefit
from interaction with stromal cells during Ara-C or VP-16 exposure (Figure 1C). This suggests the presence of intracellular
pathways that are unique to B-lineage leukemic cells and that can
be regulated by interaction with the bone marrow microenviron-
ment. However, significant components appear common to Blineage ALL cells because JM-1, RS4, and SUP-B15 were uniformly protected from chemotherapy-induced cell death by stromal
cell coculture, in spite of phenotypic differences (Figure 1).
The role of stromal cells in protecting tumor cells from
chemotherapy has clinical and biologic implications. In addition to
leukemic cells, epithelial tumors, including breast29 and prostate30
cancer, have been shown to metastasize to the bone marrow. As do
leukemic cells, residual tumor cells in these diseases that escape
treatment contribute to relapse. For that reason, it will be essential
to evaluate the effect of stromal cells on chemotherapeutic
regimens used for various tumors. Understanding the role of
stromal cells in the maintenance of minimal residual disease in
hematopoietic and epithelial tumors is essential for the design of
improved therapies to eradicate affected cells. This is particularly
important because of the aggressive phenotype of tumor cells at
relapse,31 which contributes to disease that is often refractory to
standard treatment and ultimately to poor clinical outcome.
Acknowledgments
The authors thank Drs Kenneth S. Landreth and Solveig G. Ericson
for critically reviewing this manuscript.
References
1. Goulden N, Langlands K, Steward C, et al. PCR
assessment of bone marrow status in “isolated”
extramedullary relapse of childhood B-precursor
acute lymphoblastic leukaemia. Br J Haematol.
1994;87:282-285.
tion of spatial organization and antigenicity of leukocyte surface molecules by aldehyde fixation:
flow cytometry and high-resolution FESEM studies of CD62L, CD11b, and Thy-1. J Histochem
Cytochem. 1996;44:1115-1122.
2. Miniero R, Saracco P, Pastore G, et al. Relapse
after first cessation of therapy in childhood acute
lymphoblastic leukemia: a 10-year follow-up
study, Italian Association of Pediatric Hematology-Oncology (AIEOP). Med Pediatr Oncol.
1995;24:71-76.
13. Wilmanns C, Fan D, O’Brian CA, Bucana CD,
Fidler IJ. Orthotopic and ectopic organ environments differentially influence the sensitivity of murine colon carcinoma cells to doxorubicin and
5-fluorouracil. Int J Cancer. 1992;52:98-104.
3. Miyake K, Medina K, Ishihara K, Kimoto M, Auerbach R, Kincade PW. A VCAM-like adhesion molecule on murine bone marrow stromal cells mediates binding of lymphocyte precursors in culture.
J Cell Biol. 1991;114:557-565.
4. Ryan DH. Adherence of normal and neoplastic
human B cell precursors to the bone marrow microenvironment. Blood Cells. 1993;19:225-241.
5. Kinashi T, Springer TA. Adhesion molecules in
hematopoietic cells. Blood Cells. 1994;20:25-44.
6. Kincade PW. Cell interaction molecules and cytokines which participate in B lymphopoiesis. Baillieres Clin Haematol. 1992;5:575-598.
7. Makrynikola V, Kabral A, Bradstock KF. Effects of
recombinant human cytokines on precursor-B
acute lymphoblastic leukemia cells. Exp Hematol.
1991;19:674-679.
8. Manabe A, Coustan-Smith E, Behm FG, Raimondi SC, Campana D. Bone marrow-derived
stromal cells prevent apoptotic cell death in Blineage acute lymphoblastic leukemia. Blood.
1992;79:2370-2377.
9. Gibson LF, Fortney J, Landreth KS, Piktel D, Ericson SG, Lynch JP. Disruption of bone marrow
stromal cell function by etoposide. Biol Blood
Marrow Transplant. 1997;3:122-132.
10. Haskell CM, Berek JS. Cancer Treatment, 4th ed.
Philadelphia: WB Saunders; 1995.
11. Roberts RA, Spooncer E, Parkinson EK, Lord BI,
Allen TD, Dexter TM. Metabolically inactive 3T3
cells can substitute for marrow stromal cells to
promote the proliferation and development of
multipotent haemopoietic stem cells. J Cell
Physiol. 1987;132:203-214.
12. Hasslen SR, Burns AR, Simon SI, et al. Preserva-
14. Staroselsky AN, Fan D, O’Brian CA, Bucana CD,
Gupta KP, Fidler IJ. Site-dependent differences in
response of the UV-2237 murine fibrosarcoma to
systemic therapy with adriamycin. Cancer Res.
1990;50:7775-7780.
15. Murti KG, Brown PS, Kumagai M, Campana D.
Molecular interactions between human B-cell progenitors and the bone marrow microenvironment.
Exp Cell Res. 1996;226:47-58.
16. Van d V, Smits AJ, Verdaasdonk MA, et al. ␤1Integrins dominate cell traffic of leukemic cells in
human bone- marrow stroma. Int J Cancer. 1996;
66:225-233.
17. Bradstock K, Makrynikola V, Bianchi A, Byth K.
Analysis of the mechanism of adhesion of precursor-B acute lymphoblastic leukemia cells to bone
marrow fibroblasts. Blood. 1993;82:3437-3444.
18. Ryan DH, Nuccie BL, Abboud CN, Winslow JM.
Vascular cell adhesion molecule-1 and the integrin VLA-4 mediate adhesion of human B cell
precursors to cultured bone marrow adherent
cells. J Clin Invest. 1991;88:995-1004.
19. Filshie R, Gottlieb D, Bradstock K. VLA-4 is involved
in the engraftment of the human pre-B acute lymphoblastic leukaemia cell line NALM-6 in SCID mice.
Br J Haematol. 1998;102:1292-1300.
20. Brisco MJ, Condon J, Hughes E, et al. Outcome
prediction in childhood acute lymphoblastic leukaemia by molecular quantification of residual
disease at the end of induction [see comments].
Lancet. 1994;343:196-200.
21. Shah N, Oseth L, LeBien TW. Development of a
model for evaluating the interaction between human pre-B acute lymphoblastic leukemic cells
and the bone marrow stromal cell microenvironment. Blood. 1998;92:3817-3828.
22. Gibson LF, Piktel D, Narayanan R, Nunez G, Landreth KS. Stromal cells regulate bcl-2 and bax
expression in pro-B cells. Exp Hematol. 1996;24:
628-637.
23. Miyashita T, Reed JC. Bcl-2 oncoprotein blocks
chemotherapy-induced apoptosis in a human leukemia cell line. Blood. 1993;81:151-157.
24. Thomas A, El Rouby S, Reed JC, et al. Drug-induced apoptosis in B-cell chronic lymphocytic
leukemia: relationship between p53 gene mutation and bcl-2/bax proteins in drug resistance.
Oncogene. 1996;12:1055-1062.
25. McConkey DJ, Chandra J, Wright S, et al. Apoptosis sensitivity in chronic lymphocytic leukemia is
determined by endogenous endonuclease content and relative expression of BCL-2 and BAX.
J Immunol. 1996;156:2624-2630.
26. Taylor ST, Hickman JA, Dive C. Epigenetic determinants of resistance to etoposide regulation of
Bcl- X(L. and Bax by tumor microenvironmental
factors [see comments]. J Natl Cancer Inst. 2000;
92:18-23.
27. Jiang H, Sugimoto K, Sawada H, et al. Mutual
education between hematopoietic cells and bone
marrow stromal cells through direct cell-to-cell
contact: factors that determine the growth of bone
marrow stroma-dependent leukemic (HB-1) cells.
Blood. 1998;92:834-841.
28. Elices MJ, Osborn L, Takada Y, et al. VCAM-1
on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the
VLA-4/fibronectin binding site. Cell. 1990;60:
577-584.
29. Menard S, Squicciarini P, Luini A, et al. Immunodetection of bone marrow micrometastases in
breast carcinoma patients and its correlation with
primary tumour prognostic features. Br J Cancer.
1994;69:1126-1129.
30. Mansi JL, Berger U, Wilson P, Shearer R,
Coombes RC. Detection of tumor cells in bone
marrow of patients with prostatic carcinoma by
immunocytochemical techniques. J Urol. 1988;
139:545-548.
31. Kamel-Reid S, Letarte M, Doedens M, et al. Bone
marrow from children in relapse with pre-B acute lymphoblastic leukemia proliferates and disseminates
rapidly in scid mice. Blood. 1991;78:2973-2981.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
2000 96: 1926-1932
Stromal cells regulate survival of B-lineage leukemic cells during
chemotherapy
Ryan E. Mudry, James E. Fortney, Teresa York, Brett M. Hall and Laura F. Gibson
Updated information and services can be found at:
http://www.bloodjournal.org/content/96/5/1926.full.html
Articles on similar topics can be found in the following Blood collections
Apoptosis (747 articles)
Neoplasia (4182 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society
of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.