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Prepublished online as a Blood First Edition Paper on June 7, 2002; DOI 10.1182/blood-2002-01-0030.
NEOPLASIA
From the Virginia Piper Cancer Institute, Abbott
Northwestern Hospital, Minneapolis, MN; and Department of Biomedical
Engineering, Lerner Research Institute, The Cleveland Clinic
Foundation, OH.
Hyaluronan (HA) is suggested to play a role in the pathophysiology
of multiple myeloma. To further investigate the role of HA in this
disease, we examined hyaluronan synthase (Has) gene expression and HA
production in bone marrow mesenchymal progenitor cells (bmMPCs) derived
from multiple myeloma patients. The relative abundance of mRNA for each
HAS gene was determined using competitive reverse
transcription-polymerase chain reaction (cRT-PCR), whereas HA
production was detected by fluorophore-assisted carbohydrate electrophoresis (FACE). We determined the basal expression of Has
isoforms in myeloma bmMPCs and then compared this expression with
expression in healthy donor bmMPCs. Of the 3 Has isoforms, Has1 mRNA was expressed predominantly in myeloma bmMPCs, with expression 7.6-fold greater than Has2. Compared with normal bmMPCs, Has1 mRNA expression was 20-fold greater in myeloma bmMPCs. Normal bmMPCs predominantly expressed Has2 mRNA (8.2-fold greater than myeloma
bmMPCs). Upon coculture of myeloma bmMPCs with plasma cells, Has1
transcript was strongly attenuated. FACE results show that myeloma
bmMPCs synthesize 5.7-fold more HA than those from healthy
donors. These data suggest that myeloma bmMPCs could be an important
component of the myeloma pathophysiology in vivo by their increased
expression of extracellular matrix (ECM) components relevant to plasma
cell growth and survival.
(Blood. 2002;100:2578-2585) Hyaluronan, a major constituent of the
extracellular matrix (ECM), is a large nonprotein
glycosaminoglycan composed of repeating units of glucuronic acid and
N-acetylglucosamine. Hyaluronan has been shown to play an
important role in matrix assembly, cell proliferation, cell migration,
and embryonic/tissue development.1-4 There is increasing
evidence that hyaluronan plays an important role in tumorigenesis and
in the process of tumor angiogenesis.5 In particular,
hyaluronan production is up-regulated in a variety of malignant tissues
compared with normal cells, and increased hyaluronan levels have been
shown to correlate with increased tumor cell invasion, migration, and
proliferation.6-8
A role for hyaluronan in multiple myeloma pathophysiology has
recently been suggested, where abnormally high or low serum hyaluronan
has been correlated with poor prognosis,9,10 conveying the
prognostic importance of this molecule. Multiple myeloma is a
malignancy characterized by the accumulation of malignant plasma cells
within the bone marrow, wherein soluble factors and cell-cell contact
likely provide optimal conditions for proliferation and survival. In
vitro coculture systems have shown that in addition to the bone marrow
environment providing cell-cell contact and the elaboration of growth
factors/interleukins, interactions with bone marrow ECM likely affect
the proliferation and survival of the myeloma cells and their response
to chemotherapeutic agents.11-13 The bone marrow ECM
consists primarily of fibronectin, collagen types I and IV, laminin,
and the glycosaminoglycans heparan sulfate, chondroitin sulfate, and
hyaluronan.14 Myeloma cells express multiple adhesion
receptors, including the hyaluronan receptors CD44 and RHAMM (receptor
for hyaluronan-mediated motility).15-17 Interaction of hyaluronan with hyaluronan receptors initiates intracellular signal-mediated events, including cell adhesion, migration, proliferation, and protection of myeloma cells from apoptosis.18-20 Clinically, the expression of the v9
isoform of CD44 by myeloma plasma cells is related to decreased overall
patient survival.21,22
The overall production of hyaluronan is determined by the enzymatic
activities of hyaluronan synthase and hyaluronidase. Three hyaluronan
synthase (Has) genes have been described, each encoding a
plasma membrane protein responsible for hyaluronan
synthesis.23-25 Has genes are differentially expressed
during embryonic development, and Has1, Has2, and Has3 have been
identified in various cell types, among them adult peripheral blood
lymphocytes. Although expression of any one HAS gene is sufficient for
hyaluronan synthesis, the contribution of the 3 HAS gene products to
hyaluronan production and expression is not yet clear.
As a first step toward discerning the role of hyaluronan as an integral
component of the bone marrow extracellular matrix in multiple myeloma
patients, we have characterized Has gene expression and hyaluronan
production in bone marrow mesenchymal progenitor cells (bmMPCs) derived
from multiple myeloma and healthy donors. We show that myeloma
bmMPCs predominantly express message for Has1 and furthermore
synthesize more hyaluronan than do healthy donors. Because
myeloma plasma cells reside within the bone marrow in close proximity
with cellular and extracellular components of the bone marrow
microenvironment, these data support the idea that further
characterization of the bone marrow compartment is required to assess
its role in the pathophysiology of multiple myeloma.
Cell lines and myeloma plasma cells
Reagents
Isolation and growth of bmMPCs Fresh bone marrow aspirate samples were obtained from newly diagnosed myeloma patients (n = 13) upon receiving informed consent. Fresh bone marrow aspirate samples were obtained from age-matched healthy donors (n = 14) with no prior history of malignancy, after informed consent was obtained. Bone marrow aspirates were harvested into preservative-free heparin.BmMPCs were isolated and expanded by incubating isolated bone marrow
mononuclear cells at a concentration of 1 × 106/mL in 5 mL Oligonucleotide primers Sense and antisense primers were prepared by Integrated DNA Technologies (IDT, Coralville, IA). Has primers were derived outside the conserved coding sequences to ensure specific amplification of Has1, Has2, and Has3. -Actin was used for normalization of cDNA as
well as for evaluation of RNA integrity. Bands obtained by PCR
amplification with each primer set were excised, purified from the
agarose gel, sequenced, and the resultant sequence was compared with
each HAS gene sequence. Each primer set was found to specifically
amplify Has1, Has2, and Has3 only. The primers used are indicated in
Table 1.
Reverse transcription-polymerase chain reaction Total RNA was extracted using Trizol reagent (Gibco) from bmMPC cultures at no greater than 80% confluence. Extraction was performed following the manufacturer's protocol. The RNA pellet was resuspended in 20 µL DEPC-treated water and the RNA concentration was determined. RNA was stored at 70°C until ready for use.
Reverse transcription was performed using total RNA under the following
conditions: 1.0 µg total RNA, 1 × reaction buffer, 5 mM
MgCl2, 1 mmol deoxyribonucleoside triphosphates (dNTPs), 1.6 µg oligo-p(dT)18 primer, 40 units RNase inhibitor,
and 20 units avian myeloblastosis virus (AMV) reverse transcriptase
(Seikagaku America) to a total volume of 20 µL. The
samples were incubated at 25°C for 10 minutes, followed by 42°C for
60 minutes. PCR was used to detect the presence of Has1, Has2, and
Has3. Competitive reverse transcription coupled with polymerase chain reaction Construction of the recombinant Has DNA to be used as the internal standard (Has competitor) used primers containing sequences for a portion of-actin mRNA.28 The 3' end of the
forward primer contained 20 bases complementary to the -actin gene,
with the remaining 39 bases containing Has 5' sequence and T7 promoter sequence. The reverse primer contains reverse -actin complementary sequence at the 3' end as well as the reverse Has sequence and a
poly(dT)18 sequence. These primers were used to
amplify the recombinant internal standard (Has competitor) from normal
bone marrow cDNA. The PCR reaction was performed as follows: 1 µL
cDNA, 1 × PCR buffer, 2 mM MgCL2, 0.5 µM dNTPs, 0.8 µM Has competitive RT-PCR (cRT-PCR) 5' insert primer, 0.8 µM Has
cRT-PCR 3' insert primer, and 2.5 units Taq polymerase (Qiagen).
Cycling conditions were 1 cycle at 95°C for 5 minutes and 49°C for
45 seconds; 72°C for 1 minute; 2 cycles at 95°C for 1 minute,
49°C for 45 seconds, and 72°C for 1 minute; 30 cycles at 95°C for
1 minute, 58°C for 45 seconds, and 72°C for 1 minute; and 1 cycle
at 95°C for 1 minute, 58°C for 45 seconds, and 72°C for 10 minutes. The internal standard PCR fragment was gel purified
using the QIAquick gel extraction kit (Qiagen), eluted in 20 µL Tris
(tris(hydroxymethyl)aminomethane)-HCl (pH 8.5), and used
directly for in vitro transcription. The transcription reaction was
performed using the Riboquant kit (Pharmingen). The insert was purified
by phenol-chloroform extraction and ethanol precipitation and
resuspended in DEPC-treated water. The recombinant RNA concentrations
were determined by spectrophotometry. Serial dilutions were made at 10 pg/µL, 1 pg/µL, 0.1 pg/µL, 0.01 pg/µL, 0.001 pg/µL, 0.0001 pg/µL, 0.000 01 pg/µL, 0.000 001 pg/µL, and 0.000 0001 pg/µL
and used in cRT-PCR.
The cRT-PCR was performed with 1 µg total RNA and recombinant RNA at concentrations indicated above. RT was performed in a total volume of 5 µL containing 0.4 µg oligo-p(dT)18 primer, 1 × reaction buffer, 1.25 mmol MgCl2, 0.25 mmol dNTPs, 10 units RNase inhibitor, and 5 units AMV and incubated at 25°C for 10 minutes followed by 42°C for 60 minutes. The entire volume of generated cDNA was then used immediately for PCR. PCR was performed in a 25 µL total volume containing 1 × PCR buffer, 2 mmol MgCl2, 0.3 mmol dNTPs, 60 pmol primer, and 1.25 units Taq polymerase; with 30 cycles at 94°C for 5 minutes, 30 seconds at 94°C, 30 seconds at 60°C, and 30 seconds at 68°C; followed by 7 minutes 68°C. The PCR products were visualized on a 1.5% agarose gel stained with ethidium bromide. To determine copy number, the cDNA prepared from 1.0 µg total RNA was kept constant and the competitor concentration was decreased as indicated above in log dilutions. The PCR products were scanned by densitometry, and the scanning units were normalized to the size of the competitor. The densitometry units were plotted against the number of copies of the competitor template. The copy numbers for Has transcripts in myeloma and normal bmMPCs are the crossover points at which the competitor and the bmMPC cDNA gave equal amounts of PCR product. Coculture experiments BmMPCs were established, as indicated above, in 24-well plates. When cultures reached 80% confluency, the medium of each was aspirated, and 1 × 105 plasma cells in -MEM, 10% FBS
were added to each well. BmMPCs and plasma cells were in coculture for
1, 6, and 12 hours at 37°C, 5% CO2, followed by
treatment with 0.01% trypsin for 2 minutes to remove any adherent
plasma cells. After trypsin treatment, bmMPC monolayers were rinsed 3 times in 2.0 mL PBS, followed by lysis in Trizol reagent, and used for
RT-PCR as indicated above. Removal of the plasma cells and purity of
the bmMPCs after trypsinization were determined by flow cytometry.
Purity of bmMPCs and removal of plasma cells were determined by the
presence of cells expressing CD45 as measured by flow cytometry.
Immunofluorescence Analysis of surface marker expression on bmMPCs was performed by single-color immunofluorescence. Briefly, cells were stained with an antibody conjugated to FITC for 60 minutes at 4°C, followed by 2 washes in PBS containing 10% FBS and 0.02% azide. Samples were analyzed on a FACSCalibur (Becton Dickinson). Files of 10 000 events were collected. Staining with a specific mAb is compared with staining with the appropriate isotype-matched control in all cases.Proteinase K digestion of culture media and cell layer fractions At the end of the culture period (48 hours after medium change), each medium fraction (about 5 mL) was transferred to a pretared tube and stored at20°C. Culture flasks containing the cell layers were
stored at 20°C. A 2.5 mg/mL stock solution of proteinase K (PK) was
made fresh in 0.0005% phenol red, 100 mmol ammonium acetate, pH 7.0 (digest buffer); a 250 µL aliquot of the stock solution added to each
medium fraction; and the samples digested for 2 hours at 60°C with
mixing every 30 minutes. The PK stock solution was diluted 1:10 (250 µg/mL final concentration) with digest buffer, 3 mL added to each
cell layer, and the samples digested for 2 hours at 60°C with mixing
every 30 minutes. A second 250 µL aliquot of a fresh PK stock
solution was added to each medium sample prior to digestion for an
additional 2 hours at 60°C with mixing every 30 minutes. A second 3 mL aliquot of a 1:10 dilution of fresh PK stock solution was added to
each cell layer sample prior to digestion for an additional 2 hours at
60°C with mixing every 30 minutes. The cell layer PK digests were
transferred from each culture flask to a pretared tube, and each flask
was rinsed twice with 2 mL digest buffer. Rinses were combined with their corresponding PK digests for each cell layer sample. Both the
medium and cell layer samples were then heated at 90°C for 10 minutes
to inactivate the PK. The medium fractions were adjusted to 5.5 mL by
addition of digest buffer; 5 mL culture medium was processed as
described above and served as a control for any preexisting hyaluronan
and/or chondroitin sulfate present in the FBS supplement.
Double ethanol precipitation All of each PK-digested cell layer sample (about 10 mL) and one fifth of each PK-digested medium sample (1.1 mL) were transferred to pretared tubes and concentrated on a vacuum concentrator to 300 µL as determined by weight (1 µL = 1 mg). For the cell/matrix fractions this required multiple transfer and concentration steps. A total of 1.0 mL of20°C absolute ethanol was added to the sample concentrate
(about 77% final ethanol concentration). The samples were mixed
thoroughly and incubated overnight at 20°C. The samples were
centrifuged at 10 000g for 15 minutes at 4°C to pellet
macromolecular material including hyaluronan. The supernatant fractions
were aspirated to waste. Each pellet was washed with 1 mL of 20°C absolute ethanol and centrifuged at 10 000g for 15 minutes
at 4°C. The pellet wash was aspirated to waste. The precipitate was dried and resuspended in 300 µL digest buffer. A total of 1.0 mL of
20°C absolute ethanol was added to each resuspended pellet and the
samples were precipitated a second time as described above. The
supernatant and pellet wash from each sample were aspirated to waste.
The precipitate fractions were resuspended in 100 or 200 µL digest
buffer prior to enzymatic digestion.
Enzymatic digestion A total of 100 µL of each medium and precipitate fraction was treated as follows: 1 aliquot was digested for 1 hour at 37°C, with 100 mU/mL hyaluronidase SD, followed by 1 hour at 37°C with 100 mU/mL chondroitinase ABC and 2 hours at 37°C with 0.5 U/mL glucoamylase. Where applicable, an aliquot was left untreated with any enzyme activity. The samples were frozen on dry ice and lyophilized prior to derivatization as described below.Fluorescent derivatization with 2-aminoacridone, fluorophore-assisted carbohydrate electrophoresis, gel imaging, and data analysis All samples were derivatized as previously described29 by addition of 40 µL of 12.5 mmol AMAC (500 nmol) in 85% DMSO/15% acetic acid followed by incubation for 15 minutes at room temperature. A total of 40 µL of 1.25 M sodium cyanoborohydride (50 000 nmol) in ultrapure water was added followed by incubation for 16 hours at 37°C. After derivatization, 20 µL glycerol (20% final concentration) was added to each sample prior to electrophoresis.A total of 5 µL (1:20) of each derivatization reaction was electrophoresed using MONO composition gels with MONO gel running buffer as previously described.29 The gels were illuminated with UV light (365 nm) from an Ultra Lum Transilluminator and imaged with a Quantix cooled CCD camera from Roper Scientific/Photometrics with the specifications previously described.30 The images were analyzed by Gel-Pro Analyzer 3.0 (Media Cybernetics). Digital images for each gel were taken at 2 exposures: one with oversaturated pixel intensity to allow visualization of less abundant derivatized structures and a second exposure with pixels within a linear 12-bit depth range that was used for quantitation. Baseline was determined using the "join valleys" method set at 1%. Statistical analysis of HAS expression For each HAS isoform, we made 2 comparisons. Among normal and myeloma bmMPCs, we compared the fold difference in relative transcript levels of Has1 versus Has2, respectively. We also compared the fold difference in relative transcript levels of Has1 versus Has2 between normal and myeloma bmMPCs. Within each patient population, relative amounts of Has1 and Has2 were transformed on the log scale and then compared using a paired t test. The difference in the relative amounts of Has1 and Has2, also calculated on the log scale, was then compared between patient populations using the Student t test.
Differential expression of Has mRNA in bone marrow mesenchymal progenitor cells We examined Has isoform expression in bmMPC cultures derived from normal and multiple myeloma donors by RT-PCR. The primers used to detect each HAS isoform are indicated in Table 1 and were derived from the coding sequences of Has1, Has2, and Has3. PCR products for Has1 and Has2 were detected in all normal and myeloma bmMPCs examined (Figure 1A). However, using 2 separate sets of RT-PCR primers, Has3 product was only variably detected in normal donor bmMPCs and detected in only 1 of 7 multiple myeloma patient bmMPC cultures (data not shown). These same primer sets were able to amplify Has2 and Has3 from 4 myeloma plasma cell lines. Similarly, Has2 but not Has1 was expressed in CD38+ bone marrow plasma cells from a myeloma patient; however, Has3 was not detectable (Figure 1A). The lack of detectable Has3 in CD38+ bone marrow plasma cells was further confirmed in CD38+ bone marrow plasma cells from 3 additional patients (data not shown). Based on the weak and/or no expression of Has3 in both myeloma bmMPCs and bone marrow CD38+ plasma cells, we focused our further analysis of HAS expression to Has1 and Has2.
For quantification of Has1 and Has2 mRNA expression, competitive
quantitative RT-PCR was performed. Figure 1B indicates the amplification of Has1 and Has2 mRNA in 3 individual healthy
donor and multiple myeloma-derived bmMPC cultures. The amount of
competitor in the PCR ranged from 0.1 × 102 to
1 × 10
Normal donor bmMPCs predominantly expressed Has2 mRNA, with expression 21.5-fold greater than Has1 (9.1 × 104 copies per microgram RNA Has2 and 2.3 × 103 copies per microgram RNA Has1). Has2 mRNA in normal bmMPCs was 8.2-fold greater than Has2 mRNA in myeloma bmMPCs (9.1 × 104 copies per microgram RNA in normal bmMPCs and 6.1 × 103 copies per microgram RNA in myeloma MPCs) (Figure 1B, Table 2). Coculture of myeloma plasma cells modulates bmMPC Has expression Because myeloma plasma cells reside within the bone marrow in juxtaposition with stromal cells, we examined the consequence of plasma cell contact on HAS expression in myeloma bmMPCs. By RT-PCR we noted that of 4 plasma cell lines examined, all expressed mRNA for Has2 and Has3 but not for Has1 (Figure 1A). The presence of Has2 and lack of Has1 mRNA observed in the plasma cell lines is identical to the expression pattern observed in ex vivo bone marrow plasma cells (Figure 1A). Due to the difficulty in obtaining sufficient numbers of purified bone marrow plasma cells to carry out the coculture experiments, we chose ANBL-6 (IL-6 dependent) and ARH77 (IL-6 independent) myeloma plasma cell lines to coculture with myeloma bmMPCs for 1, 6, and 12 hours. Both ANBL-6 and ARH77 cells were in direct contact with bmMPCs during the time course assay. A total of 35% ± 8% of ARH77 cells and 30% ± 5% of ANBL-6 cells were found to adhere to bmMPC monolayers by 1 hour, and this percentage did not change over the time course of coculture (data not shown). After the coculture period, nonadherent and adherent cells were removed as described in "Materials and methods," and Has expression was determined. Removal of adherent plasma cells was determined by assessing the presence of CD45+ cells, which is expressed by bone marrow plasma cells but not by bmMPCs. As shown in Figure 2, no CD45+ cells remained in the bmMPC monolayer after trypsinization at either the 1-hour or 12-hour time points.
Upon coculture with ARH77 cells, Has1 mRNA expression was reduced by 80% ± 5% in bmMPCs derived from 2 individual myeloma donors within the first 6 hours of coculture and remained at this level after 12 hours of coculture with ARH77 cells (Figure 2). In the second myeloma MPC culture, Has1 expression was reduced to less than 5% by 12 hours of coculture with ARH77 (Figure 2). In contrast to Has1 expression, Has2 mRNA expression in myeloma bmMPCs was unaltered after 12 hours of coculture (data not shown). Down-regulation of Has1 was also observed upon coculture of myeloma bmMPCs with the ANBL-6 plasma cell line, where Has1 expression was reduced by 40% as early as 1 hour after coculture, to less than 5% by 6 hours, and remained at this level at 12 hours of coculture (Figure 2). As observed with ARH77 coculture, Has2 mRNA expression in bmMPCs remained unaltered (data not shown). Fluorophore-assisted carbohydrate electrophoretic analysis of hyaluronan in bmMPC cultures FACE was used to measure the relative amounts of HA present in normal and myeloma bmMPC cultures. BmMPCs were also cultured with a corticosteroid (Dex), which has been shown to reduce HA expression in other cell culture models.31 In this study, HA synthesis is determined through measurement of AMAC-derivatized DiHA, the
smallest characteristic repeat saccharide for HA generated by
hyaluronidase SD digestion (unsaturated glucuronic acid residue at the
nonreducing terminal linked to N-acetylglucosamine).
Most of the HA synthesized by bmMPCs was secreted into the culture
medium with all of the HA partitioning into the medium ethanol
precipitate fraction (Figure 3). No HA
was detected in fresh control medium, which was never used to culture
cells (data not shown); therefore, all of the HA detected as
AMAC-derivatized
Although most of the HA synthesized by the bmMPCs in culture was
secreted into the culture medium (Figure 3), some HA was retained in
the cell layer fraction. As shown in Figure
4, the cell layer ethanol precipitate
fractions contain all of the HA present in the cell layers. Unlike in
the medium samples in Figure 3, in the absence of Dex, the amount of HA
present in the cell layer from the bmMPCs of myeloma patients was
slightly lower than that in healthy donors. However, similar
to the medium samples in Figure 3, the amounts of HA in the cell layers
were reduced in the presence of Dex. In the absence of enzyme
digestion, no AMAC-derivatized glucose or
glycosaminoglycan-related bands were observed (data not shown) similar
to those seen in Figure 3. An unknown AMAC-derivatized saccharide (X1)
is present in only the myeloma patient-derived bmMPCs. The
AMAC-derivatized glucose in the samples in Figure 4 is derived from
cellular macromolecular glycogen recovered in the precipitate fraction
and depolymerized by the action of glucoamylase. There is approximately
10-fold more glycogen in the myeloma cultures than in the normal
cultures. As shown in Figure 4B, the AMAC-derivatized glucose bands in
lanes 6 and 7 were covered to allow imaging of the AMAC-derivatized
HA, a ubiquitous component of the ECM, is synthesized by a class of membrane-bound HAS proteins.23-25 There is increasing evidence that HA production is up-regulated in tumors and may play a significant role in tumor progression.5-8 Results from this study suggest that HA expression within bone marrow mesenchymal progenitor cells derived from myeloma patients is increased. To examine HA synthesis in bmMPCs from multiple myeloma patients, we have examined the expression of HAS mRNA, specifically Has1 and Has2 in bmMPCs by quantitative RT-PCR and HA synthesis by FACE analysis. We have found that myeloma bmMPCs express predominantly Has 1 and synthesize 5- to 10-fold greater HA than do normal bmMPCs. Within the bone marrow microenvironment, cell-cell contact and elaboration of growth factors and cytokines are important in the maintenance of normal homeostasis and hematopoiesis. In multiple myeloma, the bone marrow microenvironment is likewise believed to play an important role, providing favorable signals for plasma cell growth and survival. In particular, IL-6 produced by the bone marrow stromal cells is a major survival and proliferation factor for myeloma cells.32-34 We have shown that IL-6 and other cytokines and growth factors are up-regulated in myeloma bone marrow mesenchymal progenitor cells in the absence of plasma cell contact, indicating an abnormality within the myeloma bone marrow environment that may impact plasma cell growth and survival. It has been previously shown that myeloma bone marrow stromal cells exhibit a simpler deposition of ECM proteins such as fibronectin, laminin, and collagen type IV compared with that observed on normal donor bone marrow stromal cells.35 The data presented in this paper provide further and novel evidence of differences within the HA component of bone marrow ECM in myeloma compared with healthy donors. The clinical implications of altered bone marrow ECM on myeloma disease progression are not yet clear. FACE analysis of the HA synthesized by bmMPCs in culture showed that bmMPCs derived from myeloma patients synthesized 5- to 10-fold more HA in the absence of dexamethasone than bmMPCs from healthy donors. In the presence of dexamethasone, the amount of HA synthesized by bmMPCs derived from healthy donors and myeloma patients was reduced to approximately a third of initial values, but the HA synthesized by the bmMPCs from the multiple myeloma patients remained 10-fold higher than that for the bmMPCs from healthy donors. There was a differential retention of HA in the cell layers of bmMPC cultures from healthy donors and multiple myeloma patients, with less retained when cultured with dexamethasone. Because most of the HA is secreted into the medium, the cell
layer results may reflect differential contamination of the cell layers
with their respective culture medium. However, if this were true, the
ratio of Among the 3 HAS isoforms examined, Has1 transcript levels were up-regulated to the greatest extent in myeloma bmMPCs compared with normal donor bmMPCs. Although our studies did not determine the molecular weight of HA produced in bmMPCs, the increase in Has1 transcript did correspond to an observed increase in HA production. Each HAS protein can independently catalyze HA synthesis, although each HAS protein has different enzymatic properties. Has3 has been shown to be more active and produce a shorter HA chain than either Has1 or Has2, whereas Has1 and Has2 produce HA chain lengths of similar molecular weight. The various HA chain lengths may in turn impact different cell functions such as proliferation and migration,4 although to date differences in biologic function have been observed in vitro comparing small oligosaccharide chains of HA (30-50 kDa in size) with large HA chains of 1 million to 3 million kDa. Our observations indicate a switch between Has2 and Has1 in myeloma bmMPCs that likely will not reflect a difference in overall chain size but may reflect differences in overall function or deposition of HA within the bone marrow extracellular matrix and may have important implications for tissue remodeling. Cytokine and growth factors have been shown to modulate both HAS
message and HA synthesis, where differential regulation of HAS
transcripts and cell type-specific regulation in response to factors
have been observed. Transforming growth factor-
We are grateful to the staff of the Biomedical Imaging and Processing Laboratory and Flow Cytometry Core Facility (University of Minnesota) and to Robin Bliss (BioStatistics Core, University of Minnesota Cancer Center) for expert technical assistance and advice. We thank Aniq Darr, Hyunjin Rho, Alicia Felthauser, Stephanie Wallace, and Nancy Gin for their expert technical assistance. We thank the staff of the TwinCities Spine Center (Minneapolis, MN) and the University of Minnesota Cancer Center Tissue Procurement Facility for provision of normal donor bone marrow.
Submitted January 4, 2002; accepted May 13, 2002.
Prepublished online as Blood First Edition Paper, June 7, 2002; DOI 10.1182/blood-2002-01-0030.
Supported in part by the Cleveland Clinic Foundation and by grant support from the Allina Medical Foundation.
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.
Reprints: Anna M. Masellis, Virginia Piper Cancer Institute-39419, Abbott Northwestern Hospital, 800 E 28th St, Minneapolis, MN 55407; e-mail: anna.masellis{at}allina.com.
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12.
Hazlehurst L-A, Damiano J-S, Buyuksal I, Pledger W-J, Dalton W-S.
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Abstract
Introduction
6/L), which
was added at the initiation of plating MPC cultures.
