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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 4 (February 15), 1999:
pp. 1287-1298
Muc-1 Core Protein Is Expressed on Multiple Myeloma Cells and Is
Induced by Dexamethasone
By
Steven P. Treon,
Joseph A. Mollick,
Mitsuyoshi Urashima,
Gerrard Teoh,
Dharminder Chauhan,
Atsushi Ogata,
Noopur Raje,
Joseph H.M. Hilgers,
Lee Nadler,
Andrew R. Belch,
Linda M. Pilarski, and
Kenneth
C. Anderson
From the Department of Adult Oncology, Dana Farber Cancer Institute,
and Department of Medicine, Harvard Medical School, Boston, MA; the
Division of Hematology and Oncology, Massachusetts General Hospital,
Boston, MA; the Department of Haematology, Singapore General Hospital,
Singapore; the Academisch Ziekenhuis, Vrije Universiteit, Amsterdam,
The Netherlands; and the Cross Cancer Institute, University of Alberta,
Edmonton, Alberta, Canada.
 |
ABSTRACT |
Monoclonal antibodies (MoAbs) that selectively identify Muc-1 core
protein (MoAbs DF3-P, VU-4H5) determinants were used to identify the
Muc-1 glycoform present on 7 multiple myeloma (MM) cell lines, 5 MM
patient plasma cells, 12 MM patient B cells, as well as 32 non-MM cell
lines and normal hematopoietic cells. Flow cytometry studies
demonstrated that all MM cell lines, MM patient plasma cells, and MM
patient B cells expressed Muc-1 core protein epitopes. Circulating B
cells from 4 normal donors also expressed Muc-1 core protein. In
contrast, Muc-1 core protein was absent on 28 of 32 non-MM neoplastic
cell lines, 17 of which expressed Muc-1. Splenic and tonsillar B cells,
CD34+ stem cells, resting T cells, and bone marrow plasma
cells obtained from normal donors both lacked Muc-1 glycoforms. We next
studied the effects of estrogen, progesterone, and glucocorticoid
receptor agonists and antagonists on Muc-1 expression, because
consensus sequences for the response elements of these steroids are
present on the Muc-1 gene promoter. These studies showed that
dexamethasone (Dex) induced Muc-1 expression on MM cell lines, as
determined by both flow cytometry and Western blot analyses. Dex also
induced upregulation of Muc-1 on prostate and ovarian cancer cell
lines. Time and dose-response studies demonstrated that Dex induced
maximal cell surface Muc-1 expression by 24 hours at concentrations of 10 8 mol/L. Dex induced Muc-1 upregulation could be
blocked with a 10-fold excess of the glucocorticoid receptor antagonist
RU486, confirming that Dex was acting via the glucocorticoid receptor. No changes in Muc-1 expression were observed on MM cells treated with
estrogen and progesterone receptor agonists and antagonists or with
RU486. These studies provide the framework for targeting Muc-1 core
protein in vaccination and serotherapy trials in MM. In addition, the
finding that Muc-1 expression on MM cells can be augmented by Dex at
pharmacologically achievable levels suggests their potential utility in
enhancing treatments targeting Muc-1 in MM.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ALTHOUGH MULTIPLE myeloma (MM) is
sensitive to chemotherapy with a median survival 3 to 4 years, most
patients are not cured and eventually succumb to MM. However, MM cells
express both patient-specific (ie, idiotype) and myeloma-associated
antigens that are ideal targets for immunotherapy. One such
MM-associated antigen is the core protein of MUC-1 (polymorphic
epithelial mucin, epithelial membrane antigen, DF3
antigen).1 Muc-1 is normally present on the luminal surface
of secretory glands as a highly glycosylated transmembrane protein. Its
extracellular domain is made up of 20 amino acid tandem repeats, with a
variable number (range, 21 to 125) of copies within one Muc-1
molecule.2 The variable number of tandem repeat (VNTR)
domain of Muc-1 is immunogenic: most of the 56 monoclonal antibodies
(MoAbs) submitted to the TD-4 workshop bound to epitopes within the
VNTR domain.3,4 The specificity of antibody binding to the
VNTR domain depends largely on the extent of glycosylation of
Muc-1.5-7 In contrast to normal glandular tissues,
adenocarcinomas of the breast, ovary, pancreas, and lung express Muc-1
in an aberrant, hypoglycosylated form.1 Hypoglycosylated
variants of Muc-1 appear to arise from mutations in glycosylation
enzymes, resulting in the addition of shorter and less branched
carbohydrate chains to the Muc-1 core protein backbone.8
These differences in the glycosylation patterns of Muc-1 have permitted
the identification of MoAbs that distinguish its various
glycoforms.3-7
Muc-1 is defined as an epithelial antigen. It is also expressed on MM
cell lines, fresh patient MM cells, and plasmacytomas,9-13 as well as other hematological malignancies, including Ki-1-positive B-cell lymphomas, T-cell lymphomas, Hodgkin's disease, and malignant histiocytosis.9,10,14-16 The function of Muc-1 is not fully understood. Potential roles as an
immunostimulatory12,17,18 or as an
immunoinhibitory19,20 molecule have been proposed. Furthermore, Muc-1 binds to intercellular adhesion molecule-1 (ICAM-1),
suggesting a role in cellular migration.21 Moreover, previous studies have identified consensus sequences for steroid response elements for estrogen, progesterone, and glucocorticoids on
the Muc-1 promoter.22,23 Although a regulatory
role for ovarian steroids on MUC-1 expression in mouse24
and baboon25 uterus has been reported, no studies have yet
examined the effect of steroids on MUC-1 expression on human cells.
In the present studies, we characterized the glycoform expression of
Muc-1 on freshly obtained MM patient plasma cells and MM cell lines, as
well as on normal donor plasma cells. Moreover, we examined Muc-1
glycoform expression on MM cells, as well as on hematologic and
carcinoma cell lines. Because the MM clone extends from pre-B
cells,26 sIgM+ preswitched B
cells,27,28 to late stage B cells,29,30 we also
studied Muc-1 glycoform expression on B cells from MM patients as well
as normal donors. Given the steroid response elements on the Muc-1
promoter, we also examined the effect of treatment with estrogen,
progesterone, and glucocorticoid receptor agonists and antagonists on
Muc-1 expression on the cell surface of human MM cells. These studies
demonstrate that MM plasma cells and B cells express Muc-1 core protein
determinants and that cell surface expression of Muc-1 can be
upregulated by dexamethasone. Our data therefore suggest the potential
utility of incorporating glucocorticoids into immunotherapeutic
treatment strategies targeting Muc-1 in MM.
| |
MATERIALS AND METHODS |
Cell lines and culture conditions.
ARH-77, CESS, CMK, K562, MCF.7, RPMI 8226, U266, and ZR-75 cell lines
were obtained from the American Type Culture Collection (Rockville,
MD). OCI My-5 cells31 were kindly provided by Dr H.A.
Messner (Ontario Cancer Institute, Toronto, Ontario,
Canada); interleukin-6 (IL-6)-transfected S6B45 MM
cells32 were kindly provided by Dr T. Kishimoto (Osaka
University, Osaka, Japan). Jurkat, B-15, NALM 6, HZ, DHL6, DHL16, and
RL cell lines were provided by Dr Lee Nadler (Dana-Farber
Cancer Institute, Boston, MA). L5174T, SW480, HUTU80, HS294T, and AC5
cell lines were obtained from Dr Selwyn Broitman (Boston University
Medical School, Boston, MA). AGSB gastric, T.Tu esophageal, and CAPAN-2
pancreatic cancer cells were provided by Drs Robert Hennihan, Anil
Rustgi, and Andrew Warshaw, respectively (Massachusetts General
Hospital, Boston, MA). 36M, CAOV-3, and OVC3 ovarian cancer cells were
obtained from Dr Stephen Cannistra (Dana-Farber Cancer Institute).
Lastly, DU145 and PC3 prostate cancer cells were kindly provided by Dr Arthur Pardee (Dana-Farber Cancer Institute). All MM (except as noted
below), acute lymphocytic, non-Hodgkin's lymphoma (NHL), lymphoblastic, leukemic, megakaryocytic, prostatic, and 786-0 renal
cancer cell lines were cultured in RPMI 1640 medium (GIBCO, Grand
Island, NY). OCI-My5 MM cells were cultured in Iscove's Modified
Dulbecco's Medium (GIBCO). Breast, colon, duodenal, esophageal, gastric, melanoma, ovarian, A458, and ACHN renal cancer cell lines were
cultured in Dulbecco's Modified Eagle's Medium. CAPAN-2 pancreatic and Caki-1 renal cancer cells were cultured in McCoy's 5A medium. All
media contained 10% fetal bovine serum (FBS; Hyclone, Logan, UT) and
was supplemented with L-glutamine, penicillin, and streptomycin (GIBCO).
Patients.
Peripheral blood (PB) and/or bone marrow (BM) was obtained from
17 patients with MM after informed consent was obtained. MM patients
included those at diagnosis, during intermittent chemotherapy, in
stable phase, and in relapse. Peripheral blood was obtained from 6 healthy normal volunteer donors, and BM was obtained from 4 donors with
nonmalignant disease, termed normal for the purpose of this study.
Antibodies.
DF3 and DF3-P MoAbs were the kind gift of Dr Donald Kufe (Dana-Farber
Cancer Institute). The antigenic determinants for these antibodies have
been previously characterized.7 VU-3C6 and VU-4H5 MoAbs
were purified at our institution from hybridomas kindly provided by Dr
J. Hilgers (Vrije Universiteit, Amsterdam, The
Netherlands); the epitopes targeted by these antibodies have been
extensively characterized.3,4 DF3 and VU-3C6 MoAbs bind to
both Muc-1 glycosylated and core protein epitopes, whereas DF3-P and
VU-4H5 MoAbs specifically bind to core protein epitopes within the VNTR
domain of Muc-1.3,4 B4-FITC (CD19) MoAb was obtained from
Coulter (Hialeah, FL). FMC63 (CD19) MoAb33 was conjugated
to fluorescein isothiocyanate (FITC). Anti-CD38 (Leu17-phycoerythrin [PE]) MoAb was purchased from Becton Dickinson (San Jose, CA). FMC44
(CD45RA) MoAb34 was conjugated to FITC or PE and antihuman Ig (goat antihuman Ig [H+L]-FITC), and goat antimouse Ig-PE were purchased from Southern Biotechnology (Birmingham, AL).
Phenotypic analyses of cell lines and immunofluorescence.
MM and other neoplastic cell lines were characterized by flow cytometry
for cell surface Muc-1 expression. Cells (106) were
incubated with either anti-Muc-1 or control MoAbs (5.0 µg) for 45 minutes. Cells were then washed with phosphate-buffered saline (PBS)
and incubated for 45 minutes with 2.0 µg of goat antimouse
fluorescein-conjugated MoAb (Coulter). Cells were next washed and fixed
with 1% formaldehyde PBS. Staining intensities were defined by
comparing differences in log fluorescence for anti-Muc-1 MoAbs versus
isotype control MoAbs, using the following criteria: (0) no difference;
(+) <1 log fold difference ; (++)  1 but < 2 log difference; and
(+++) 2 log difference.
Phenotypic analysis of patient cells and immunofluorescence.
PB or BM aspirates were collected into heparinized vacutainer tubes and
centrifuged over a ficoll-hypaque gradient (Pharmacia, Uppsala,
Sweden). To identify plasma cells from patients or normal donors, BM mononuclear cells (MC) were stained in three-color immunofluorescence using CD38-FITC and CD45RA-PE together with Muc-1
MoAbs or isotype-matched control MoAbs (indirect immunofluorescence). Files were gated for CD38hi45RA cells
and the staining for Muc-1 epitopes plotted as a histogram as compared
with an identically gated sample stained with an isotype-matched control MoAb. For PBMC samples, cells were stained in two-color immunofluorescence using CD19-FITC and Muc-1 or isotype-matched control
MoAb (indirect immunofluorescence). The number of CD19+ B
cells in MM patients and normal donors is identical when detected using
either FMC63 or with B4 MoAb. Files were gated for CD19 cells and side
scatter, and the staining for Muc-1 epitopes was plotted as a histogram
as compared with identically gated isotype-matched control samples. For
MM PBMC, CD19+ B cells as defined here have been previously
shown to express CD19 and IgH transcripts with an IgH VDJ sequence
identical to that of autologous BM plasma cells.33,35,36
Normal T cells were isolated from PBMC using the E-Rosette
technique.37 The purity of isolated T cells was confirmed
by flow cytometric analysis for CD3 expression. CD34+ cells
were isolated from the PB of healthy donors using Ceprate CD34 columns
(Cell Pro, Bothell, WA).38
Preparation of steroid agonists and antagonists.
17 estradiol (E2), tamoxifen (TAM), progesterone (PRG), RU486, and
water-soluble dexamethasone (DEX) were obtained from Sigma Chemical (St
Louis, MO). ICI 182,780 (ICI) was the kind gift from Zeneca
Pharmaceuticals (Wilmington, DE). Stock solutions
(102 mol/L in absolute alcohol) of E2, TAM, PRG, and
RU486 were stored at  20°C. Water-soluble Dex was solubilized
in PBS at a 102 mol/L stock solution and stored at
4°C. Cells were cultured for 24 to 72 hours with varying
concentrations of steroids, which were freshly prepared for each experiment.
Immunoblotting assays for MUC-1.
Cell extracts from MM cell lines (RPMI 8226 and S6B45), breast cancer
cell lines (MCF.7 and ZR-75), and prostatic cancer cells (DU145) were
examined by Western blot analysis. Cells were lysed by incubation for
30 minutes in ice-cold lysis buffer (50 mmol/L Tris-HCl [pH 8.0], 150 mmol/L NaCl, 0.1% Nonidet P-40, 1 mmol/L EDTA, 5 µg/mL phenylmethyl
sulfonyl fluoride [PMSF], 1 mmol/L Na3VO4, 2 µg/mL aprotinin, 2 µg/mL leupeptin, and 50 mmol/L NaF). Aliquots of
lysates were electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein was then
transferred onto nitrocellulose membranes, and nonspecific binding was
blocked by overnight incubation with 5% skim milk. Subsequently, the
membranes were washed with TBS-Tween and incubated with either VU-3C6,
VU-4H5, DF3, or DF3-P MoAbs (5 µg; diluted 1:200). DMA1
anti- -tubulin MoAb (1 µg; diluted 1:1,000) was used as a control
antibody. An enhanced chemiluminescence (ECL) kit (Amersham, Arlington
Heights, IL) was used to visualize bound antibodies.
| |
RESULTS |
Expression of Muc-1 core protein on MM and other neoplastic cell
lines.
In these studies, Muc-1 expression on 7 human MM cell lines, along with
32 other neoplastic cell lines, was defined by staining with MoAbs that
bind Muc-1 glycosylated and core protein epitopes (VU-3C6, DF3) as well
as MoAbs binding specifically to Muc-1 core protein (VU-4H5, DF3-P) in
flow cytometric analysis. These studies demonstrated that 7 of 7 MM
lines (ARH-77, HS Sultan, IM9, OCI My-5, RPMI 8226, S6B45, and U266)
expressed Muc-1 on their cell surface, as evidenced by reactivity with
the VU-3C6 MoAb. The intensities of Muc-1 expression using the VU-3C6
MoAb were as follows: low (U266), medium (ARH-77, HS Sultan, IM9), and
high (OCI My-5, RPMI 8226, S6B45) (Fig 1
and Table 1). These results paralleled
those obtained with the DF3 MoAb, which identified Muc-1 on 6 of 7 MM
lines (Table 1). In particular, DF3 staining was absent on HS Sultan
cells, but was present on the remainder of MM cell lines with the
following staining patterns: low (ARH-77, IM9) and high (OCI My-5, RPMI
8226, S6B45, U266) (Table 1). Muc-1 was also identified by either
VU-3C6 or DF3 MoAbs on 17 of 32 non-MM neoplastic cell lines: Jurkat
ALL; HZ NHL; MCF.7 and ZR-75 breast; CESS B-cell lymphoblasts; SW480
colon; T.Tu esophageal; CMK megakaryocytic; 36M, CAOV-3 and OVC3
ovarian; Capan-2 pancreatic; DU145 and PC3 prostatic; as well as A498,
ACHN, and Caki-1 renal cancer cells. The intensities of Muc-1 staining
among non-MM, Muc-1-positive cells was strongest on the breast,
esophageal, ovarian, pancreatic, prostatic, and renal cancer cell lines
(Table 1).
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| Fig 1.
Expression of Muc-1 on human MM and breast cancer cell
lines. Human MM (ARH-77, OCI My-5, RPMI 8226, S6B45 and U266) and
breast cancer (ZR-75) cell lines were examined by flow cytometry for
cell surface expression of Muc-1 glycosylated protein by staining with
the VU-3C6 MoAb relative to an isotype control MoAb.
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Interestingly, expression of Muc-1 core protein, determined by
reactivity with the VU-4H5 and DF3 MoAbs, was more selective for MM
cell lines (Fig 2 and Table 1). Using the
VU-4H5 MoAb, Muc-1 core protein was detected on the cell surface of 7 of 7 MM cell lines with the following staining intensities: low
(ARH-77, HS Sultan, OCI My-5, U266) and medium (IM9, RPMI 8226, S6B45) (Table 1). In addition, Muc-1 core protein was identified on 4 of 7 MM
cell lines using the DF3-P MoAb with the following patterns of
reactivity: low (U266, OCI My-5, S6B45) and medium (RPMI 8226) (Table
1). In contrast to MM cells, 28 of 32 (88%) of non-MM neoplastic cell
lines, including MCF.7 and 2R-75 breast as well as OVC.3 and 36M
ovarian cell lines, lacked Muc-1 core protein, as determined by
reactivity with the VU-4H5 or DF3-P MoAbs. Three of 4 non-MM cell lines
that expressed Muc-1 core protein were of B-cell lineage, akin to the
origin of MM cells, whereas the fourth cell line was CAPAN-2, which is
a pancreatic cancer cell line. Expression of Muc-1 core protein in
these 4 non-MM cell lines was of low intensity. As can be seen in Table
1, expression of Muc-1 core protein was absent on breast, esophageal,
ovarian, prostatic, and renal cancer cell lines, all of which expressed Muc-1 by use of the VU-3C6 and DF3 MoAbs.
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| Fig 2.
Expression of Muc-1 core protein on human MM, breast
cancer, and ovarian cancer cell lines. Human MM (ARH-77, OCI My-5, RPMI
8226, and S6B45), breast cancer (MCF.7 and ZR-75), and ovarian cancer
(OVC.3 and 36M) cell lines were examined by flow cytometry for cell
surface expression of Muc-1 core protein by staining with the VU-4H5
MoAb relative to an isotype control MoAb.
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Expression of Muc-1 core protein on plasma cells from MM patients and
normal donors.
To assess Muc-1 expression on freshly isolated plasma cells from MM
patients, BMMC from 5 MM patients were stained in multicolor immunofluorescence using CD38, CD45RA, and MoAbs that detect both Muc-1
glycosylated and core protein epitopes (VU-3C6) or specifically identify core protein (VU-4H5). Of CD38hi
45RA plasma cells from 5 of 5 MM patients, 55% ± 12% (range, 13% to 78%) express Muc-1 core protein
(Fig 3 and
Table 2). By use of the VU-3C6 MoAb, Muc-1
epitopes were identified on 38% ± 13% (range, 8% to 84%) of MM
plasma cells from these 5 MM patients. In contrast, for 4 of 4 donors,
normal plasma cells had very low or no expression of any Muc-1
glycoform using either MoAb (Fig 3 and Table 2).
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| Fig 3.
Expression of Muc-1 core protein on MM patient and normal
donor plasma cells. BM plasma cells from 2 representative MM patients
(A) and 2 representative normal donors (B) are shown. Files were gated
for CD38hi45RA plasma cells (15% to 87%
and 1% to 5% were present in MM patients and normal donors,
respectively). Muc-1 on plasma cells was plotted as a histogram, with
bold lines indicating staining with the VU-4H5 or VU-3C6 anti-Muc-1
MoAbs and the thin lines representing staining by an identically gated
isotype-matched control MoAb.
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Expression of Muc-1 core protein on B cells from MM patients and
normal donors.
Previous work has shown that circulating B cells in MM patients are
part of the malignant clone as defined by their expression of
clonotypic IgH transcripts.33,35,36 PB B cells from 12 of
12 MM patients were analyzed for expression of Muc-1 epitopes (Fig 4A and Table 2). B cells were defined
as CD19+ cells, including those cells with a high side
scatter (SSchi).27,36 The SSchi set
of MM B cells has been shown to be almost exclusively clonotypic, whereas the SSclow set includes both clonotypic and
polyclonal B cells.33,35 The majority of CD19+
B cells in MM patients (85% ± 3%) expressed Muc-1 core protein (Fig 4A and Table 2). Both the SSchi and SSclow
sets of MM B cells were predominantly Muc-1+ (not shown).
Polyclonal B cells (49% ± 10%) from 4 of 4 normal donors, defined
as CD19+SSclow PBMC, also expressed Muc-1 core
protein (Fig 4B and Table 2). In contrast to normal PB B cells, splenic
and tonsillar B cells from 3 of 3 normal donors lacked Muc-1, by virtue
of lack of reactivity with the VU-4H5 and VU-3C6 MoAbs (data not
shown).
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| Fig 4.
Expression of Muc-1 core protein on MM patient and normal
donor B cells. Peripheral blood B cells from 2 representative MM
patients (A) and 2 representative normal donors (B) are shown, with
Muc-1 staining plotted as a histogram. The bold line shows Muc-1
staining on B cells, and the thin line shows isotype-matched control
staining on an identically gated aliquot of cells. Shown are B cells
(26% to 32% and 4% to 7% for MM patients and normal donors,
respectively), which include SSchi gated PB B cells for MM
patients, because these are clonotypic, having IgH and CD19
transcripts.32,34,35 In contrast, SSchi PB B
cells are excluded from the B-cell gate in normal donors, because these
are monocytes.35
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Expression of Muc-1 on normal CD34+ hematopoietic
progenitor cells and T cells.
We next examined whether Muc-1 was expressed on CD34+
hematopoietic progenitor cells and on resting T cells obtained from 3 normal donors using the VU-4H5 and VU-3C6 MoAbs. Neither PB-derived CD34+ hematopoietic progenitor cells nor resting T cells
from normal donors express Muc-1 (data not shown).
Effects of estrogen, progesterone, and glucocorticoid receptor
agonists and antagonists action on Muc-1 expression.
Although consensus sequences of steroid response elements for estrogen,
progesterone, and glucocorticoid receptors have been identified on the
promoter of the Muc-1 gene,21,26 no functional analyses of
these steroid response elements has been reported in human cells. We
therefore treated S6B45, RPMI 8226, and OCI My-5 MM cells; MCF.7 and
ZR-75 breast cancer cells; CAOV-3 and OVC-3 ovarian cancer cells; and
DU145 prostate cancer cells with agonists and antagonists for estrogen
(17 estradiol, diethylstilbestrol, tamoxifen, and ICI 182,780),
progesterone (progesterone, RU486), and/or glucocorticoid (Dex,
RU486) receptors and assayed for related changes in Muc-1 cell surface
expression. Dex induced upregulation of Muc-1, as evidenced by
reactivity with the VU-4H5, DF3-P, VU-3C6, and DF3 MoAbs, using flow
cytometry and/or Western blot analyses of S6B45 and RPMI 8226 (Figs 5 and 6),
of OCI My-5 MM cells, of CAOV-3 and OVC-3 ovarian cancer cells, and of
DU145 prostate cancer cells (Figs 5 and 6). Dex induction of Muc-1
expression was most pronounced (1 log fold increase in fluorescence
intensity) on S6B45 and RPMI 8226 MM cells and on DU145 prostatic
cancer cells (Fig 5). Dose-response studies using Dex
(1012 to 106 mol/L) showed
maximal induction of cell surface Muc-1 expression on S6B45 MM
(Fig 7) and DU145 prostatic cancer cells at
108 mol/L. Dex induction of Muc-1 expression on
S6B45, RPMI 8226, and OCI My-5 MM cells, as well as on DU145 prostate
cancer cells was also evaluated at several intervals of Dex treatment
(24, 48, and 72 hours) and shown to be maximal at 24 hours (data not shown). As can be seen in Fig 8, Dex
induction of Muc-1 expression on two MM cell lines (S6B45 and RPMI
8226) was blocked by 10-fold excess of the glucocorticoid receptor
antagonist RU486, consistent with a glucocorticoid receptor-mediated
mechanism of Muc-1 induction by Dex.
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| Fig 5.
Effect of Dex on cell surface Muc-1 expression. RPMI 8226 MM cells, S6B45 MM cells, and DU145 prostate cancer cells were cultured
in media alone or with Dex (108 mol/L) for 24 hours.
Muc-1 expression was assessed by flow cytometry by staining with the
VU-4H5 and VU-3C6 MoAbs relative to isotype control MoAbs. Peaks shown
are denoted as follows: (*) staining with isotype control MoAb; (**)
staining of cells cultured in media alone using either the VU-4H5 or
the VU-3C6 MoAbs; (***) staining of cells cultured with Dex using
either the VU-4H5 or the VU-3C6 MoAbs. No change in isotype control
MoAb staining was seen with Dex stimulation.
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| Fig 6.
Immunoblotting assays for Muc-1 expression in human MM
and prostate cancer cell lines stimulated with Dex. Cell lysates
obtained from human MM (S6B45 and RPMI 8226) and prostate cancer
(DU145) cells cultured with media alone or with Dex (108
mol/L) for 24 hours were examined by Western blot analysis using the
VU-4H5 MoAb. Dual bands seen in the lysates from S6B45 and DU145 cells
show coallelic expression of Muc-1.1 The DMA1 MoAb to
 -tubulin was used as a control antibody.
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| Fig 7.
Dose-response relationship for Dex induction of Muc-1
expression on S6B45 MM cells. S6B45 MM cells were cultured for 24 hours
with Dex (1012 to 106 mol/L) and analyzed
by flow cytometry for Muc-1 expression using VU-4H5, VU-3C6 and isotype
control MoAbs. *Cells stained with isotype control. **Cells cultured in
media alone and stained with either the VU-4H5 or the VU-3C6 MoAbs.
***Cells cultured with Dex and stained with either the VU-4H5 or the
VU-3C6 MoAbs.
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| Fig 8.
Effect of RU486 on Dex induction of Muc-1 expression on
MM cells. S6B45 and RPMI 8226 MM cells were cultured in media alone or
with Dex (108 mol/L) in the presence or absence of the
glucocorticoid receptor antagonist RU486. (A) Changes in Muc-1
expression after culturing with Dex. (B) Changes in Muc-1 expression
after culturing with both Dex and RU486. (C) Changes in isotype control
MoAb staining in media alone, Dex alone, and Dex plus RU486 cultures.
(a) Cells stained with isotype control MoAb. (b) Cells cultured with
media alone and stained with the VU-4H5 MoAb. (c) Cells cultured with
Dex and stained with the VU-4H5 MoAb. (d) Cells cultured with both Dex
and RU486 and stained with the VU-4H5 MoAb.
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No changes in Muc-1 expression were observed on MCF.7 and ZR-75 breast
cancer cell lines treated with Dex, as assessed by both FACS and
Western blot analyses (data not shown). In addition, treatment of
S6B45, RPMI 8226, and OCI My-5 MM cells; MCF.7 and 2R75 breast cancer
cells; and DU145 prostate cancer cells with estrogen and progesterone
receptor agonists and antagonists, or alone with the glucocorticoid
receptor antagonist RU486, did not alter Muc-1 cell surface expression
(data not shown).
| |
DISCUSSION |
The use of immunotherapy for the treatment of MM has previously
targeted cell surface antigens on MM cells such as CD16, CD38, CD54,
and HM1.24.39-45 Unfortunately, these antigens are present (CD16, CD38, CD54) or have not been fully examined (HM1.24) on normal
tissues. In these studies, we have identified Muc-1 core protein as a
selective target for MM-directed immunotherapy. Muc-1 is a suitable
target for immunotherapy, because epitopes that give rise to antibody
and cytotoxic T-lymphocyte reactivity may be repeated 21 to 125 times
within one molecule of Muc-1 due to genetic polymorphisms.1
Both antibody46,47 and cytotoxic T-cell
responses13,17,18 specific to Muc-1 have been observed in
patients with Muc-1-bearing malignancies, and these responses can be
elicited by vaccination with Muc-1 conjugated to mannan or keyhole
limpet hemocyanin (KLH).48,49 The latter studies are of a
phase I nature and show that Muc-1 vaccinations appear to be well
tolerated.49 Although no clinical response data have so far
been generated from those cancer patients immunized with Muc-1
vaccines, considerable preclinical data using tumor rejection models
have shown that Muc-1-directed vaccinations are biologically active.
Specifically, vaccinations against Muc-1 using Muc-1
cDNA,50 recombinant vaccinia virus containing Muc-1 gene
sequences,51 Muc-1-KLH conjugated protein,52 or
fusion cells made up of dendritic cells and Muc-1-bearing
tumors53 can prevent establishment and dissemination of
inoculated Muc-1-bearing tumors, as well as induce rejection of
established Muc-1-positive tumors in mice. Muc-1 may also serve as a
target for serotherapy.54,55 Athymic mice xenografted with
Muc-1-bearing ovarian tumors show preferential tumor uptake of
I125-conjugated DF3 MoAb.54 Moreover, the
anti-Muc-1 MoAb HMFG1 conjugated to yttrium-90 has been used in phase
I/II clinical trials involving ovarian cancer patients, with benefit
suggested when used in an adjuvant setting in patients with no evident
disease after surgery.55
The presence of Muc-1 on the cell surface of MM cells has previously
been identified by several investigators.9-13 However, these studies did not identify the Muc-1 glycoform present on MM cells.
Because glycosylation patterns of Muc-1 vary among normal and malignant
tissues, knowledge of the Muc-1 glycoform present on MM cells may be
important in identifying selective epitopes to serve as targets for
immunotherapy. In these studies, we have demonstrated that Muc-1 core
protein is more selectively expressed on MM cell lines, on patient MM
plasma cells and B cells, and on certain (ie, circulating) normal donor
B cells. In contrast, Muc-1 core protein is absent on 28 of 32 (88%)
non-MM neoplastic cell lines, including breast, esophageal, prostate,
ovarian, and renal cancer cell lines that express Muc-1 in a
glycosylated form. Moreover, a survey of CD34+ cells,
resting T cells, splenic and tonsillar B cells, and plasma cells from
normal donors did not show cell surface expression of Muc-1 core
protein. Lastly, by immunohistochemistry normal human small intestine
(VU-4H5), colon (VU-4H5), and breast (DF3-P, VU-4H5) tissues do not
show reactivity when stained with the anti-Muc-1 core protein MoAbs
used in these studies.7,56 Taken together, the
above-described studies demonstrate that MM plasma and B cells, as well
as circulating normal B cells, selectively express Muc-1 core protein
on their cell surface. The finding that circulating MM B cells, in
addition to plasma cells, express Muc-1 core protein is of particular
relevance for Muc-1-directed immunotherapy strategies for MM, because
MM B cells may be both clonotypic and act as a reservoir of
chemotherapy resistance.26-30,35
The significance of Muc-1 expression on MM plasma and B cells, along
with circulating normal B cells, remains unclear at this time. A role
in the migration of Muc-1-bearing malignant cells has previously been
suggested, because Muc-1 binds to ICAM-1.21 Such binding
may make it possible for Muc-1-bearing MM plasma cells and B cells to
adhere to endothelium and bone marrow stroma expressing
ICAM-121,57 or possibly another yet to be defined ligand(s). MM cells are known to make cytokines, including IL-1 and
tumor necrosis factor- (TNF-),58,59
which are potent inducers of ICAM-1 expression60,61 and may
stimulate ICAM-1 on endothelial and bone marrow stromal cells.
Moreover, Kaposi's sarcoma-associated herpesvirus (KSHV), which may
play a role in the pathogenesis of MM by infecting BM stromal
cells,62 may lead to cytokine elaboration and induction of
ICAM-1 expression on stromal cells, analogous to that seen in Kaposi's
sarcoma.61,63 In turn, binding of MM cells via Muc-1 to
ICAM-1 on endothelial and BM stroma cells may lead to the release of
IL-6, an important growth factor for MM cells.64 In
addition, a role for Muc-1 in mediating evasion from immune systems has
been suggested by several studies, wherein Muc-1 may either act through
stearic hindrance to evade contact with immune cells or induce anergy through a yet to be defined mechanism.1,19,20 In such
circumstances, malignant plasma cells that express Muc-1 would be able
to escape immunosurveillance, unlike normal plasma cells that, in these studies, lacked Muc-1 expression.
Because Muc-1 may have a role in immunoregulation, as well as cell
migration and adhesion, we were interested in identifying compounds
that modulate Muc-1 expression. In particular, the presence of
consensus sequences for several steroid response elements (estrogen, progesterone, and glucocorticoids)22,23 on the Muc-1
promoter suggested that steroids might alter Muc-1 expression. Although no functional analyses for these steroid response elements has been
reported in human cells, in vitro studies involving mice and baboons
suggest that estrogen and progesterone modulate uterine Muc-1
expression.24,25 In our study, Muc-1 expression was
upregulated after Dex treatment of MM cell lines, with maximal
stimulation occurred at 108 mol/L Dex, doses that
correspond to serum levels of Dex that are readily achievable
clinically.65 Muc-1 expression was also upregulated on
DU145 prostatic cancer cells, as well as on CAOV-3 and OVC-3 ovarian
cancer cells, demonstrating that Dex-induced upregulation of Muc-1 is
not restricted to MM cells. However, no change in Muc-1 expression was
seen on two breast cancer cell lines (MCF.7 and ZR-75) exposed to Dex,
showing selectivity of induction of Muc-1 by Dex. In contrast to murine
and baboon uterine Muc-1 studies,24,25 no changes in Muc-1
expression were triggered in MM, breast, and prostatic cancer cells
treated with estrogen and progesterone receptor agonists and
antagonists. These different sequelae may reflect species or tissue
specificity for Muc-1 regulation by estrogen or progesterone. In
addition, these animal studies involved in vivo treatment for longer
periods of time (up to 12 days) with estrogen and progesterone, whereas
our studies with cell lines involved in vitro exposure to steroids for
up to 72 hours. Hence, we cannot exclude the possibility that longer
intervals of exposure to estrogen and progesterone might influence
Muc-1 expression on MM cells.
In summary, our studies form the basis for targeting Muc-1 core protein
in vaccination and serotherapy trials for MM. The finding that Muc-1
expression can be augmented by Dex at pharmacologically achievable
levels suggests the potential utility of incorporating glucocorticoids
into immunotherapeutic strategies targeting Muc-1 in MM.
| |
FOOTNOTES |
Submitted May 26, 1998; accepted October 13, 1998.
Supported by National Institutes of Health grant CA78378, the Lauri
Strauss Leukemia Foundation and the International Myeloma Foundation, a
Young Investigator Award from the American Society of Clinical Oncology
(S.P.T.), and a grant from the Alberta Cancer Board Research Initiative Program.
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.
Address reprint requests to Kenneth C. Anderson, MD, Division of Adult
Oncology, Dana Farber Cancer Institute, 44 Binney St, Boston, MA 02115;
e-mail: kenneth_anderson{at}dfci.harvard.edu.
| |
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A. Moore, Z. Medarova, A. Potthast, and G. Dai
In Vivo Targeting of Underglycosylated MUC-1 Tumor Antigen Using a Multimodal Imaging Probe
Cancer Res.,
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64(5):
1821 - 1827.
[Abstract]
[Full Text]
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X. Li, L. Wang, D.P. Nunes, R.F. Troxler, and G.D. Offner
Pro-inflammatory Cytokines Up-regulate MUC1 Gene Expression in Oral Epithelial Cells
Journal of Dental Research,
November 1, 2003;
82(11):
883 - 887.
[Abstract]
[Full Text]
[PDF]
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C. Milazzo, V. L. Reichardt, M. R. Muller, F. Grunebach, and P. Brossart
Induction of myeloma-specific cytotoxic T cells using dendritic cells transfected with tumor-derived RNA
Blood,
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[Abstract]
[Full Text]
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M. Wykes, K. P. A. MacDonald, M. Tran, R. J. Quin, P. X. Xing, S. J. Gendler, D. N. J. Hart, and M. A. McGuckin
MUC1 epithelial mucin (CD227) is expressed by activated dendritic cells
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692 - 701.
[Abstract]
[Full Text]
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A. Evangelou, M. Letarte, A. Marks, and T. J. Brown
Androgen Modulation of Adhesion and Antiadhesion Molecules in PC-3 Prostate Cancer Cells Expressing Androgen Receptor
Endocrinology,
October 1, 2002;
143(10):
3897 - 3904.
[Abstract]
[Full Text]
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G. Dotti, B. Savoldo, P. Yotnda, D. Rill, and M. K. Brenner
Transgenic expression of CD40 ligand produces an in vivo antitumor immune response against both CD40+ and CD40- plasmacytoma cells
Blood,
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100(1):
200 - 207.
[Abstract]
[Full Text]
[PDF]
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N. Mitsiades, C. S. Mitsiades, V. Poulaki, D. Chauhan, P. G. Richardson, T. Hideshima, N. Munshi, S. P. Treon, and K. C. Anderson
Biologic sequelae of nuclear factor-kappa B blockade in multiple myeloma: therapeutic applications
Blood,
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4079 - 4086.
[Abstract]
[Full Text]
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N. Mitsiades, C. S. Mitsiades, V. Poulaki, K. C. Anderson, and S. P. Treon
Intracellular regulation of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human multiple myeloma cells
Blood,
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[Abstract]
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R L Ten Berge, F G M Snijdewint, S von Mensdorff-Pouilly, R J J Poort-Keesom, J J Oudejans, J W R Meijer, R Willemze, J Hilgers, and C J L M Meijer
MUC1 (EMA) is preferentially expressed by ALK positive anaplastic large cell lymphoma, in the normally glycosylated or only partly hypoglycosylated form
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December 1, 2001;
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[Abstract]
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C. S. Mitsiades, S. P. Treon, N. Mitsiades, Y. Shima, P. Richardson, R. Schlossman, T. Hideshima, and K. C. Anderson
TRAIL/Apo2L ligand selectively induces apoptosis and overcomes drug resistance in multiple myeloma: therapeutic applications
Blood,
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[Abstract]
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S. P. Treon, P. Maimonis, D. Bua, G. Young, N. Raje, J. Mollick, D. Chauhan, Y.-T. Tai, T. Hideshima, Y. Shima, et al.
Elevated soluble MUC1 levels and decreased anti-MUC1 antibody levels in patients with multiple myeloma
Blood,
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[Abstract]
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V. G. Dyomin, N. Palanisamy, K. O. Lloyd, K. Dyomina, S. C. Jhanwar, J. Houldsworth, and R. S. K. Chaganti
MUC1 is activated in a B-cell lymphoma by the t(1;14)(q21;q32) translocation and is rearranged and amplified in B-cell lymphoma subsets
Blood,
April 15, 2000;
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2666 - 2671.
[Abstract]
[Full Text]
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S. von Mensdorff-Pouilly, A. A. Verstraeten, P. Kenemans, F. G. M. Snijdewint, A. Kok, G. J. Van Kamp, M. A. Paul, P. J. Van Diest, S. Meijer, and J. Hilgers
Survival in Early Breast Cancer Patients Is Favorably Influenced by a Natural Humoral Immune Response to Polymorphic Epithelial Mucin
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[Abstract]
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K. C. Anderson, R. A. Kyle, W. S. Dalton, T. Landowski, K. Shain, R. Jove, L. Hazlehurst, and J. Berenson
Multiple Myeloma: New Insights and Therapeutic Approaches
Hematology,
January 1, 2000;
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[Abstract]
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Top
Abstract
Introduction